Lauric Acid Production in a Glycogen-Less Strain of Synechococcus sp. PCC 7002

The cyanobacterium Synechococcus sp. Pasteur culture collection 7002 was genetically engineered to synthesize biofuel-compatible medium-chain fatty acids (FAs) during photoautotrophic growth. Expression of a heterologous lauroyl-acyl carrier protein (C12:0-ACP) thioesterase with concurrent deletion of the endogenous putative acyl-ACP synthetase led to secretion of transesterifiable C12:0 FA in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in a light-emitting diode turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wild-type (WT). Inhibition of (i) glycogen synthesis by deletion of the glgC-encoded ADP-glucose pyrophosphorylase (AGPase) and (ii) protein synthesis by nitrogen deprivation were investigated as potential mechanisms for metabolite redistribution to increase FA synthesis. Deletion of AGPase led to a 10-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (ΔglgC) was modified for C12 secretion, no increase in C12 was achieved during nutrient replete growth, and no C12 was recovered from any strain upon nitrogen deprivation under the conditions used. At steady state, the growth rate of the ΔglgC strain saturated at a lower light intensity than the WT, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ΔglgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool.

In photosynthetic eukaryotes, FAs of specific chain lengths are hydrolyzed from acyl carrier protein (ACP) by thioesterase enzymes, and the released FAs move out of the chloroplast into the cytoplasm where they are activated by coenzyme A (CoA) to facilitate transfer into higher lipids (Radakovits et al., 2010;Li et al., 2013). It has been found that many bacteria, including cyanobacteria, typically bypass the free FA (FFA) intermediate when assembling newly synthesized FA into membrane lipids (Sato and Wada, 2010;Jansson, 2012). When heterologous thioesterases are expressed in certain bacteria, most of the hydrolyzed FFA is found either in the culture medium or associated with the outside of the cell (Voelker and Davies, 1994;Ohlrogge et al., 1995;Liu et al., 2011;Ruffing and Jones, 2012;Ruffing, 2014). Though the mechanism of secretion is not established, it is known that extracellular FFA can move back across the membrane and be reincorporated into metabolism by an acyl-acyl carrier protein synthetase (AAS) (Kaczmarzyk and Fulda, 2010). If this enzyme is disrupted, FFAs remain in the medium and can separate from the aqueous cultures. In the present study, carbon distribution in the cyanobacterium Synechococcus sp. Pasteur culture collection (PCC) 7002 was modified for C12 FFA synthesis by heterologous thioesterase expression (and AAS deletion) in both wild-type (WT) and carbohydrate-deficient genetic backgrounds.
The pAQ1Ex vector was modified for knockin expression of fatB1 concurrent with deletion of the putative AAS. To construct the lauric acid secretion (LAS) module, fatB1 was placed between the vector's promoter and antibiotic selection marker via NcoI/BamHI restriction sites. Flanking sequences of the fadD gene were inserted to target the cassette for homologous recombination using F||R primer pairs (5 -3 ) 1:gttcacATGCATggctaggttcg-taatctttgggggta||gtatagGAATTCgccgaaatcatggctacaatcctacttt, 2:cat-actGTCGACgatccgaatggcggaatcttcg||gttcacGCATGCgtgctggcttttgt cacaatcttcttg (restriction enzyme recognition sequences used in plasmid construction are capitalized). Transformation was accomplished following an established protocol for homologous recombination in this organism (Xu et al., 2011). Integration of the LAS module into all genome copies was achieved by increasing spectinomycin pressure and confirmed by PCR (not shown) for complete allele segregation using the 1F and 2R primers listed above. The strain SA01 contains the LAS module in a WT background, and the strain SA13 contains this module in a carbohydrate-deficient background ( Table 1).
Liquid cell cultures were grown using a rotary shaker under constant illumination in an atmosphere of 1% CO 2 , 34°C, and 160 µmol photons m −2 s −1 (µmol m −2 s −1 hereafter) photosynthetically active radiation (PAR) in 250-mL Erlenmeyer flasks with soft caps to facilitate gas exchange (VWR, Radnor, PA, USA). Batch flask cultures were grown in quadruplicate and standardized to 2.5 mg L −1 chlorophyll a at the beginning of each experiment. Pre-cultures were similarly normalized and grown to mid-linear phase (15-25 µg mL −1 chlorophyll a), whereupon cells were concentrated by centrifugation and resuspended in fresh medium for experimental replicates, which were sampled over a time course.

CONTINUOUS CULTURE
Steady-state physiology was assayed in a photobioreactor (PBR) that maintains constant optical density (turbidostasis) over a range of light intensities delivered by 630 and 680 nm light-emitting diodes (LEDs), as described previously (Melnicki et al., 2013). For maximal light penetration and steady-state illumination, cell cultures were maintained at 0.08 OD 730 in A+ medium containing 0.9 g L −1 NH 4 Cl as the nitrogen source, and Tris was omitted as pH 7.5 was maintained independently. Cultures were held at  (610), and 170/60 (759) as incident 630/680 nm light each and (in parentheses) total spherical µmol m −2 s −1 . A linear 2π incident sensor was used to measure individual wavelengths, and total spherical illumination was reported by a 4π sensor. Absorbance scans of total cell culture were measured over a 350-900 nm spectral range using a Shimadzu BioSpec 1601 spectrophotometer (Shimadzu, Kyoto, Japan). Non-transmitted fractions of 630 and 680 nm light were calculated using transmitted light values obtained in situ from the linear sensor and normalized to total spherical irradiance as described previously (Melnicki et al., 2013).

BIOCHEMICAL ANALYSES
Chlorophyll a was measured by absolute methanol extraction of a 1-mL cell pellet and calculated as described previously (Meeks and Castenholz, 1971;Porra et al., 1989). Reducing carbohydrates (RCs) were measured as glucose equivalents by a colorimetric anthrone-sulfuric acid assay described previously (Meuser et al., 2012). Dry cell weight (DCW) of batch cultures was measured from 2 mL of liquid culture concentrated by centrifugation. The cell pellet was washed once in 1 g L −1 Tris buffer (TB), resuspended in 1 mL TB, thoroughly dried at 80°C, and the dry weight of 1 mL TB subtracted to give DCW. From PBR cultures, DCW is represented as ash-free weight from 400 mL steady-state culture concentrated by centrifugation, resuspended in distilled water, dried at 105°C, and burned at 550°C for 1 h. Ash-free weight was calculated as mass lost between drying and burning.
Organic acids (OAs) were quantified by HPLC (Surveyor Plus, Thermo Scientific, Waltham, MA, USA) using 0.45-µm filtered supernatant from −N cultures. A 25-µL sample was injected onto a 150 mm × 7.8 mm fermentation monitoring column (BioRad, Hercules, CA, USA) at 0.5 mL min −1 8 mM H 2 SO 4 eluent, 45°C column operating temperature, and 50°C refractive index (RI) detector operating temperature, in parallel with a photodiode array detector for absorbance at 210 nm. A standard mix of acetate, pyruvate, succinate, α-ketoglutarate, and α-ketoisocaproate was used for quantification, and all samples were held at 10°C in a thermostated sample tray before injection.
Fatty acyl content was measured as transesterifiable fatty acid methyl esters (FAMEs) using an adapted method . Briefly, 0.5 mL of liquid culture was hydrolyzed and lipids saponified at 100°C for 2 h in 1 mL 95:5% v/v absolute methanol:0.8 g L −1 KOH (in H 2 O), after which 1.5 mL 94.2:5.8% v/v methanol:12N HCl was added for acid-catalyzed methylation at 80°C for 5 h. FAMEs were extracted into 1 mL n-hexane and the extract was analyzed using an Agilent 7890A gas chromatograph (GC) and DB-5ms column with flame ionization detection (Agilent Technologies, Santa Clara, CA, USA). A flow rate of 1.15 mL min −1 H 2 carrier gas was used to separate FAMEs at 20°C min −1 to 230°C, held for 1 min, then 20°C min −1 to 310°C and held for 5 min. A standard mix of FAMEs was used for quantification and retention time correlation (37-component FAME mix, Supelco, Bellefonte, PA, USA). Due to insufficient resolution between unsaturated C18 FAs, the combined contents of 18:1, 18:2, and 18:3 are reported as 18:n. The unknown (unk) compound that elutes prior to C16:1 was not included in FAME tabulations. A two-tailed t -test was performed to determine statistical significance (p-value). Lauric acid methyl ester (C12 FAME) was identified via mass spectral analysis conducted using a Varian 3800 GC and Varian 1200 quadrupole MS/MS (Agilent Technologies, Santa Clara, CA, USA) equipped with a Rxi-5ms column (30 mm × 0.25 mm; 0.25 µm film thickness) (Restek Corporation, Bellefonte, PA, USA). A flow rate of 1.2 mL min −1 He carrier gas was used to separate FAMEs at 20°C min −1 from 70 to 230°C for a 1-min hold, then 20°C min −1 to 310°C for a 5-min hold. Mass spectra were obtained after electron ionization at 70 eV. Results were compared to the known mass spectrum of C12 FAME (NIST Mass Spec Data Center, and Stein, 2015).

PULSE AMPLITUDE MODULATION FLUOROMETRY
Variable chlorophyll fluorescence was measured using PAM fluorometry in a DUAL-PAM-100 system (Walz GmbH, Effeltrich, Germany) with a photodiode detector and RG665 filter (Schreiber, 1986). Red measuring light (620 nm) at the lowest power was pulsed at 1000 Hz during the dark and at 10,000 Hz during 635 nm actinic illumination at 98 µmol m −2 s −1 . From PBR cultures, 3 mL was immediately transferred to a cuvette and fluorescence induc- The effective quantum yield of PSII (YII') was measured by transient fluorescence changes between "J" and "I" states. The estimated redox status of the plastoquinone (PQ) pool was determined by the rise from "I" to "P" level, normalized to the total variable fluorescence observed over this period, and subtracted from 1 (Chylla and Whitmarsh, 1989). Relative changes in electron transport downstream of the PQ pool were measured by P >> S quenching as the drop from "P" to "S" states relative to the variable fluorescence (Serrano et al., 1981). The relative dark rate of PQ oxidation was obtained from the declining slope of post-illumination fluorescence, calculated from between 10 and 20 s after the level had peaked (Ryu et al., 2003). Dark-adapted measurements were taken after cells were held in the dark for 20 min and then acclimatized in actinic light for 90 s before induction.

BATCH CULTURE PRODUCTIVITY
Nitrogen replete and nitrogen deplete batch cultures of WT, SA01, ∆glgC, and SA13 were analyzed over 48 h for chlorophyll a, DCW, FAME, RC, and OA. Cultures of C12-secreting strains developed a layer of surfactant bubbles (Figure 1).

Chlorophyll a and dry cell weight
Bulk biomass accumulation in nutrient replete batch cultures yielded an increase in chlorophyll a content of 12-to 15-fold over 48 h ( Figure S1A in Supplementary Material), and DCW accumulated 4-to 5-fold ( Figure S1C in Supplementary Material). In nitrogen-deplete cultures, growth attenuation was suggested by unchanging chlorophyll a content and DCWs that were within the range of error relative to inoculum DCWs over the time course (Figures S1B,D in Supplementary Material).

Carbohydrates and organic acids
During nitrogen deprivation (−N), AGPase-disrupted strains accumulated substantially less RC than the WT background: over 24-48 h, RC on a culture volume basis reached 7-11% of WT levels in ∆glgC and 6-13% in SA13 (Figure 4A) Figure 4A). While WT and SA01 cultures developed yellow coloration during −N, the AGPase-disrupted cultures did not ( Figure 4B). Under the culturing conditions used, acetate was the most abundant OA secreted from the carbohydrate-deficient strains during −N, followed by succinate and α-ketoisocaproate (Figures 4C-E). Lesser concentrations of pyruvate and α-ketoglutarate were also observed (Figures 4F,G). Levels of OA secretion were not affected by the LAS modification.

STEADY-STATE PHYSIOLOGY
At stable growth rate for each indicated light intensity in the LED-PBR, cultures of Synechococcus sp. 7002 WT, SA01, and ∆glgC were analyzed for RC, DCW, FAME, O 2 evolution, doubling rate, and photophysiological characteristics. Growth rate and O 2 production measurements for the same conditions were made previously using a separate cultivar of WT Synechococcus sp. 7002 (not shown), which demonstrate the reproducibility of PBR measurements (Work, 2014).

Growth rates and O 2 evolution
Minimum stable doubling times of 3.5 h (WT), 3.8 h (SA01), and 4.6 h (∆glgC) were observed at 759 µmol m −2 s −1 in the WT background and at 462 µmol m −2 s −1 in ∆glgC ( Figure 6A). Bulk O 2 evolved by SA01 exceeded both WT and ∆glgC over the majority of light intensities tested (Figure 6B). On a per-doubling basis, ∆glgC produced O 2 at levels similar to WT and in fact surpassed WT at 396 µmol m −2 s −1 and above ( Figure 6C) despite www.frontiersin.org  diminished growth rates ( Figure 6A). The doubling rate required per unit DCW was similar between all strains ( Figure 6D). O 2 evolved by ∆glgC was comparable to WT by bulk DCW (Figure 6E) but greater on the basis of DCW-normalized growth rate ( Figure 6F). The uncoupling of O 2 evolution from growth rate in ∆glgC at high irradiance was not observed when further normalized to DCW ( Figure 6F). Despite attaining lower growth rates than WT at 264 µmol m −2 s −1 and above, SA01 exhibited consistently elevated O 2 evolution by volume (Figure 6B), growth rate ( Figure 6C), bulk DCW (Figure 6E), and DCW-normalized growth rate (Figure 6F).

Photophysiology
Photosynthetic electron transport appears to be altered by AGPase disruption (Figure 7). With increasing irradiance of the ∆glgC culture, a higher quantum yield of PSII was observed (Figure 7A), and the PQ pool became more reduced (Figure 7B) than the WT background. The rate of electron transport downstream of the PQ pool was also adversely affected by glgC disruption (Figure 7C), as less P >> S quenching occurred in this background with higher light. After dark adaptation, ∆glgC cultures exposed to 165 µmol m −2 s −1 and above exhibited more rapid rates of PQ oxidation in the dark ( Figure 7D).
The transmittance of 630 nm light by cell cultures was unaffected between strains ( Figure 7E), but ∆glgC transmitted less 680 nm light than the WT background ( Figure 7F) indicating more absorption or scattering by the strain at this wavelength.

DISCUSSION
Derived from photosynthetically fixed CO 2 , FAs secreted by genetically engineered cyanobacteria have yielded up to 197 mg L −1 FFA by Synechocystis sp. 6803 and 131 mg L −1 FFA (6.5 mg L −1 d −1 ) by Synechococcus sp. 7002 (Liu et al., 2011;Ruffing and Jones, 2012;Ruffing, 2014). Secretion of 4.4 mg L −1 day −1 transesterifiable lauric acid (C12) from modified strains of Synechococcus sp. 7002 was achieved in batch cultures that grew at a similar rate to WT. Sodium lauryl sulfate is a common ingredient in soap, and the foam layer atop cultures secreting C12 suggests detergent activity. Under these conditions, C12 may accumulate in surface bubbles. Phase separation may be a consideration in applying photosynthetic FA secretion on an industrial scale, and actively removing C12 from cultures, for example by hexane overlay (Davies et al., 2014) or solid-state methods (Léonard et al., 2011), may create more favorable conditions for productivity. Attenuating the synthesis of polymeric carbohydrates did not augment C12 production during normal growth, and attempts www.frontiersin.org to direct metabolism to FAs by nitrogen starvation instead eliminated C12 altogether. The absence of C12 may be due to cessation of protein and/or lipid synthesis under these conditions, or, since a phycocyanin-related promoter is responsible for fatB1 expression, the gene may be downregulated in times of nitrogen stress, as phycobiliproteins can be degraded as an intracellular nutrient source (Sauer et al., 1999;Richaud et al., 2001). Additionally, the AGPasedisrupted batch cultures exhibited a non-bleaching phenotype when nitrogen-deprived, and as previously reported, higher absorbances in the 580-650 nm phycobilin range suggest that these proteins are not deconstructed for nutrients in this background as they are in WT Davies et al., 2014). Similar characteristics were described in carbohydrate-deficient mutants of Synechocystis sp. 6803 and Synechococcus elongatus 7942 (Carrieri et al., 2012;Gründel et al., 2012;Hickman et al., 2013). Intracellular carbohydrate accumulation during nitrogen stress requires AGPase for glucose polymerization in Synechococcus sp. 7002 (Davies et al., 2014), S. elongatus PCC 7942 (Hickman et al., 2013), and Synechocystis sp. 6803 (Carrieri et al., 2012;Gründel et al., 2012), and energy spilling in the form of OA secretion was observed upon disruption of this function; and a similar outcome occurred with glycogen synthase deletions . Of the OA secreted by nitrogen-deprived ∆glgC and SA13 strains, pyruvate (C 3 ), α-ketoglutarate (C 5 ), and succinate (C 4 ) are also gluconeogenic metabolites (Zhang and Bryant, 2011;Steinhauser et al., 2012). In S. elongatus 7942, α-ketoglutarate has been demonstrated as an effector of the nitrogen regulator NtcA (Vázquez-Bermúdez et al., 2001;Tanigawa et al., 2002). Possibly derived from protein degradation or metabolite redistribution, αketoisocaproate (C 6 ) is a biosynthetic intermediate of the amino Frontiers in Bioengineering and Biotechnology | Synthetic Biology acid leucine and can be converted to acetyl-CoA and acetoacetate, which, along with pyruvate and acetate (C 2 ), are direct precursors of FAs, terpenoids, higher alcohols, and reduced storage polymers such as poly-3-hydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA). Though biosynthetic enzymes for the latter two have not been identified in Synechococcus sp. 7002 (McNeely et al., 2010), secreted OA could be supplied to a capable organism by medium exchange (Niederholtmeyer et al., 2010) or co-cultivation (Contag, 2012;Therien et al., 2014).
Steady-state photoautotrophic doubling times of 3.5 h (WT), 3.8 h (SA01), and 4.6 h (∆glgC) are close to the fastest observed in Synechococcus sp. 7002 (Ludwig and Bryant, 2012). Increased O 2 production on the basis of DCW-normalized growth rate in both SA01 and ∆glgC may represent unidentified photosynthetic energy sinks (Badger et al., 2000;Nomura et al., 2006;Suzuki et al., 2010;Zhu et al., 2010;Xu et al., 2013), which in SA01 could be related to the synthesis of secreted FA. Diminished photosynthetic productivity caused by AGPase disruption was evident, as ∆glgC reached maximum growth rate at a lower light intensity than WT. After growth rate saturation, dissipation of excess radiant energy appears to be accomplished in part by RC storage in the WT background. Restricting RC by AGPase disruption resulted in a more reduced, less oxidizable PQ pool indicating overreduction of the photosynthetic electron transport chain and/or the inability to utilize photosynthetic reductant. However, high rates of PQ oxidation by ∆glgC in the dark possibly demonstrate a respiratory or other continuous quenching function (Joët et al., 2002;Bailey et al., 2008;McDonald et al., 2011). The severity of these redox alterations may lead to the protection of PSII in ∆glgC under irradiances at which WT and SA01 accumulated RC, as evidenced by O 2 evolution decoupling, elevated PSII quantum yields, and more scattered or absorbed 680 nm light, perhaps owing in part to increased content of PSII or a phycobiliprotein such as phycocyanobilin that can absorb at 680 nm and is involved in free radical scavenging Ge et al., 2013). Demonstrating a robust capacity to manage excess light energy, Synechococcus sp. 7002 could be a promising organism in scaled systems (Dong et al., 2009;Zhu et al., 2010;Ludwig and Bryant, 2012), and efforts to reroute metabolic flux may identify enzyme targets through further investigation of carbon partitioning at high light in the present genetic backgrounds.
The planktonic cyanobacterium Synechococcus sp. 7002 was engineered to convert photosynthate into biofuel precursors, which were naturally secreted from the cell. Lauric acid and OAs can be processed into diesel and alcohols or used as a carbon source for other organisms, and their recovery from culture filtrate avoids costly cell harvesting and lysis. Though redirection of carbohydrate-deficient metabolism toward FA synthesis was not effective under the present conditions, central metabolites for FA, terpenoid, and glucan biosynthesis were generated that potentially could be captured with further metabolic adjustments for redistribution into desired pathways.

AUTHOR CONTRIBUTIONS
The LAS module was constructed by VW, who also transformed and developed the LAS strains, performed batch cultivations and biochemical assays, and compiled this manuscript. MM performed PAM fluorometry measurements and contributed to PBR research design. EH designed, built, and operated the LED-PBR to generate O 2 and dilution rate measurements, and assisted with dry weight measurements. FD performed mass spectral analysis, identified and helped quantify OAs, and assisted with quantitation of RCs. LK contributed to PAM fluorometry data collection. AB and MP were responsible for the project's conception and provided laboratory resources. All authors revised the manuscript for intellectual content.