S. elongatus Coculture for Chemical Photopr - ACS Publications

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A designed A. vinelandii-S. elongatus coculture for chemical photoproduction from air, water, phosphate and trace metals Matthew Jordan Smith, and Matthew B Francis ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00107 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 29, 2016

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ACS Synthetic Biology

A designed A. vinelandii-S. elongatus coculture for chemical photoproduction from air, water, phosphate and trace metals Matthew J. Smith and Matthew B. Francisa,b,* a

Department of Chemistry, University of California, Berkeley, California 94720-1460, and bThe Molecular Foundry, Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720-1460.

ABSTRACT: Microbial mutualisms play critical roles in a diverse number of ecosystems and have the potential to improve the efficiency of bioproduction for desirable chemicals. We investigate the growth of a photosynthetic cyanobacterium, Synechococcus elongatus PCC 7942, and a diazotroph, Azotobacter vinelandii, in coculture. From initial studies of the coculture grown in media with glutamate, we proposed a model of cross-feeding between these organisms. We then engineer a new microbial mutualism between Azotobacter vinelandii AV3 and cscB Synechococcus elongatus that grows in the absence of fixed carbon or nitrogen. The coculture cannot grow in the absence of a sucroseexporting S. elongatus, and neither organism can grow alone without fixed carbon or nitrogen. This new system has the potential to produce industrially-relevant products, such as polyhydroxybutyrate (PHB) and alginate, from air, water, phosphate, trace metals and sunlight. We demonstrate the ability of the coculture to produce PHB in this work. Keywords: Engineered coculture, metabolic syntrophy, cyanobacteria, diazotroph

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Designed cocultures have significant potential for industrial bioproductions,1-2 and several research groups have engineered systems that demonstrate this possibility. As one compelling example, Ducat et al. designed a coculture in which carbon produced by a cyanobacterium supports the heterotrophic growth of yeast,3 and in another report OrtizMarquez et al. developed a coculture in which diazotrophic nitrogen supports algal growth.4 One important goal in the field is the design of cocultures that can share both nitrogen- and carbon-containing metabolites. In nature, microorganisms have indeed evolved to allow this sharing, as exemplified by the cyanobacterium Anabaena cylindrica. The chain-like aggregates of this species use a unique “differentiation” strategy, in which some cells commit to performing nitrogen fixation.5 However, these organisms are arguably difficult to engineer to allow the production of specifically designed products. Fixed nitrogen is often considered to be the most expensive component of photosynthetic bioproductions in systems where waste water is not used as a nitrogen source.6 We propose the use of A. vinelandii as a solution to this problem in coculture systems7 because (1) it is a prodigious nitrogen fixer even under aerobic conditions, (2) it boasts a high growth rate,8 and (3) it is already capable of producing useful biopolymers. For example, it produces large amounts of the polymer alginate as a carbon starvation response as it transitions into the cyst state.9 In addition, it produces the bioplastic alternative polyhydroxybutyrate (PHB) at up to 80-90% of its biomass when a nutrient other than carbon (such as oxygen or iron) is limiting.10 Wildtype A. vinelandii is rodshaped and around 2 µm in length and secretes both ammonia4 and amino acids11 into the


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media, in addition to plant growth hormones.12,13 Alternatively, it can be readily engineered to produce increased alginate,14 to accumulate PHB during exponential growth,15 and to secrete higher levels of ammonia.16 However, this microorganism requires a fixed carbon source, which is currently considered to be cost prohibitive for the industrial production of PHB.17,18 To provide a photosynthetic carbon source for A. vinelandii, S. elongatus PCC 7942, a rod-shaped cyanobacterium around 2 µm in length, was chosen as a well-studied complementary cyanobacterial model system.19,20 Unlike many cyanobacteria, such as Nostoc and Anabaena strains, S. elongatus is naturally competent.21 Several “neutral sites” in the genome of S. elongatus PCC 7942 have been identified as locations to insert foreign genetic material,22,23 enabling it to be engineered for the secretion of several hydrophilic products.3,24 Of particular relevance to this work, it has been engineered to secrete sucrose in salt water via cloning a sucrose permease from E. coli and inserting it into the genome of S. elongatus.3 In this report, we first detail a previously unreported microbial mutualism between Azotobacter vinelandii and S. elongatus. After proposing a model of crossfeeding for the coculture, we then engineer both components of the system to allow growth in the absence of fixed carbon and nitrogen (Figure 1). While we demonstrate that the coculture grows in the absence of bicarbonate, in this work bicarbonate is added to increase the growth rate of the coculture. Using 13C labeled bicarbonate, we demonstrate that we can produce 13C-labeled PHB. This success both supports our model of syntrophy and provides a promising biosynthetic “chassis” that can be engineered further for the production of energy-rich products from air, water, trace minerals, and sunlight.



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RESULTS AND DISCUSSION We initially tested the validity of the basic cross-feeding model depicted in Figure 1A. In media containing glutamate and the trace metals necessary for both Azotobacter vinelandii and Synechoccocus elongatus growth (SAV media), it was noted that S. elongatus only grew in the presence of A. vinelandii (Figure 2, blue curve). A. vinelandii also grew to much lower optical densities when cultured alone (Figure 2, green curve). We further determined that A. vinelandii secretes ammonia into the media when grown in the SAV media under these conditions (Supporting Information Figure S1). We therefore hypothesized that the obligate photoautotroph S. elongatus was using secreted products from A. vinelandii, not glutamate, as its nitrogen source. In addition to ammonia, A. vinelandii secretes amino acids into the media when grown on a wide variety of substrates,11 which could provide additional nitrogen sources for S. elongatus. Accordingly, we tested the ability of S. elongatus to utilize several nitrogen feedstocks, including glutamate, ammonia, and several other amino acids found to be excreted by A. vinelandii.11 As shown in Supporting Information Figure S2, S. elongatus can indeed be grown using either ammonia or lysine, but not glutamate or the other amino acids tested as a nitrogen source. These results suggest that the nitrogen-containing species secreted by A. vinelandii fulfulled the nitrogen source requirements of S. elongatus in this growth media, thus confirming a key aspect of the nutrient sharing model. We next asked whether A. vinelandii also benefits from the coculture depicted in Figure 2A. While carbon fixation by communities of cyanobacteria in the ocean likely play a key role in supplying carbon to heterotrophic organisms,25 wildtype freshwater


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cyanobacteria cultured in a laboratory setting have not been shown to secrete significant amounts of carbon.3,24 Freshwater cyanobacteria have been shown to secrete small amounts of glycolate, a byproduct of photorespiration, into the supernatant.26 However, glycolate cannot be utilized by A. vinelandii.27 Interestingly, the closely related organism A. chroococcum can grow on glycolate, but the substrate is too oxidized to support nitrogen fixation.27 Based on this, we hypothesized that A. vinelandii did not benefit from S. elongatus in terms of the generation of biomass. To test this we grew the cells in dialysis bags in which the neighboring bag contained either the coculture partner or SAV media (Figure 2B). Cells in adjacent dialysis bags could share metabolities and other biomolecules, but the transfer of whole cells was prevented to allow accurate quantification of both species at the end of the experiment. The dialysis experiments demonstrated that, while S. elongatus benefited from the presence of A. vinelandii in SAV media both in terms of OD (Figure 2C) and chlorophyll synthesis (Figure 2D), A. vinelandii did not benefit from the presence of S. elongatus. Ortiz-Marquez et al. observed similar results when they cocultured microalgae with A. vinelandii.4 Taken together, these results suggested that if we engineered S. elongatus to secrete a reduced carbon source, we could obtain a mutually beneficial coculture. Ducat et al. have reported the development of an engineered cscB S. elongatus, which fixes CO2 from the atmosphere and exports sucrose in salt water.3 We engineered cscB S. elongatus in an analogous fashion, but used either an IPTG-inducible promoter or a light-activated psbA1 promoter (Supporting Information Figure S3).28 The results shown in Figures 3-5 were obtained with the psbA1 promoter, as the IPTG-inducible promoter resulted in similar coculture growth. We also redesigned the SAV media to



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contain neither fixed carbon nor nitrogen, including no citrate and no EDTA. Sodium bicarbonate was added to the media prior to use to increase the concentration of dissolved carbon dioxide and, thus, the growth rate. This modified minimal media is referred to as “CAV” herein. In SI Figure S4 we depict the growth of the coculture with and without sodium bicarbonate present. As shown in Figure 3A, wildtype A. vinelandii grown with cscB S. elongatus grows without fixed carbon or nitrogen (albeit slowly), whereas wildtype A. vinelandii with wildtype S. elongatus does not grow at all. At this point, we hypothesized that a reduced carbon source was now available to A. vinelandii, and ammonia secretion had become limiting (Figure 1B). It has been known for over a decade that deleting the nifL gene of A. vinelandii results in constituitive nitrogenase expression, and therefore the enhanced secretion of ammonia.16 This is particuarly effective due to the aerobic tolerance of the nitrogen fixing system used by this organism. Thus, to improve the efficiency of our system, we added a ∆nifL A. vinelandii (AV3)4 to the coculture and observed a significant increase in the overall growth (Figure 3A) and the chlorophyll content after 10 d in solution (Figure 3B). This further validates our model of cross-feeding, and shows the direct improvement obtained by engineering both components (Figure 1C). To quantify the increase in biomass that was derived from air, we lyophilized and weighed 10 mL portions of the AV3+cscB culture grown without sodium bicarbonate at 0 d and 6 d and saw roughly a 60% increase in mass (0 d: 91 ± 6 mg; 6 d: 145 ± 5 mg; +/SD, N=3). A control coculture comprising AV3 A. vinelandii and wildtype S. elongatus did not grow in CAV media (Figure 3A).


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Dialysis experiments were again performed to demonstrate that A. vinelandii grew to higher optical densities in CAV media going from (1) the wildtype coculture to (2) the cscB + wildtype coculture to (3) the cscB + AV3 coculture (Figure 3C). S. elongatus received only a small growth benefit in the engineered cocultures, suggesting that it grew slowly in the CAV coculture overall. In the SAV coculture (Figure 2), the S. elongatus grew at a faster rate than A. vinelandii, with the major difference between CAV and SAV media being the presence of glutamate in the SAV media. This suggests that there is not enough secreted ammonia in the engineered CAV cocultures to support robust cyanobacterial cell growth in addition to sucrose secretion and chlorophyll synthesis. We would expect that the enhanced ammonia-excreting strain of A. vinelandii would lead to a higher relative ratio of S. elongatus to A. vinelandii cells, and flow cytometry data after 24 d in CAV media, diluting 1 to 10 every 8 d, supported this conclusion (Figure 4A). After 24 d, the viable cell population for the cscB S. elongatus and AV3 A. vinelandii coculture contained roughly 10% cscB S. elongatus, which was higher than that for the cscB S. elongatus and wildtype A. vinelandii coculture (Figure 4A). Growth of the cscB S. elongatus and AV3 A. vinelandii coculture in CAV media was found to be highly dependent on both the starting cell ratio (Figure 4B and 4C) and the starting cell density (Figure 4D). In contrast, the growth of the wildtype coculture in SAV media was ultimately dependent on the amount of glutamate added to the media. Figure 3A represents the growth of the coculture with a starting cell ratio for all 4 cocultures of roughly 95:5 S. elongatus to A. vinelandii, which is far from the equilibrium value reported above (Figure 4A). Coculture growth was observed when the starting cell



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ratio was enriched in cscB S. elongatus (Figure 4B and 4C). The importance of the starting cell ratio to coculture growth supports our observations that the cscB + AV3 coculture resulted in a higher concentration of chlorophyll, but only a slight increase in S. elongatus biomass after 10 d in solution. Figure 4D demonstrates that coculture growth is affected by the number of S. elongatus cells used to initiate the coculture. This result can be explained by the dependence of the coculture on the sucrose exported by S. elongatus and the minimal growth of S. elongatus in coculture. Volumetric flow cytometry analysis supports this conclusion and enables the correlation of optical density data to increases in the number of A. vinelandii cells (SI Figures S5 and S6). Furthermore, using flow cytometry data, the generation time of A. vinelandii over 0 d to 2 d was plotted versus the starting percentage of S. elongatus. This analysis demonstrates that AV3 A. vinelandii grows faster in the presence of increasing amounts of cscB S. elongatus (SI Figure S6). We next investigated the ability of this coculture to make PHB. After 5 d of growth, PHB production as a percent of the dried coculture cell weight was 19 ± 2% (± SD, N=3). The starting optical density at 750 nm of these cocultures was 0.3 and the starting cell ratio was 90:10 S. elongatus:A.vinelandii. We used this starting cell ratio because when cscB S. elongatus outnumbers AV3 A. vinelandii, we see the highest growth rate in coculture for AV3 A. vinelandii (SI Figure S6). Azotobacter vinelandii can accumulate under some conditions between 80-90% of their dry weight as PHB.29 However, we are measuring the percentage of PHB in the coculture, and so much of the dry weight is due to non-PHB accumulating S. elongatus cells. Furthermore, optimal PHB accumulation occurs when carbon is present in excess and a nutrient such as oxygen is limiting and the only supplied carbon source we


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provided for growth was bicarbonate/carbon dioxide. For these reasons, we feel that our PHB yield is promising, though there is room for improvement. 13

C labeled bicarbonate was used to demonstrate that A. vinelandii synthesized

PHB from the sucrose secreted by S. elongatus (Figure 5). The incorporation of 13C into PHB was quantified via analyzing the four carbon containing monomer of PHB, crotonic acid, via GC-MS (SI Figure S7). Figure 5 demonstrates that the majority of the PHB found in the coculture is

13

C labeled, indicating that PHB was synthesized from carbon

fixed and then secreted by S. elongatus and not from stored carbon present in the initial amount of A. vinelandii added, for example. Only 6% of the PHB monomer contained no 13

C label and roughly 57% of the analyzed monomer contained total

13

C incorporation.

Furthermore, in Figure 5 the expected isotope pattern for crotonic acid with four labels incorporated was observed, with the 5 outcomes of the

13

13

C

C labeling of crotonic

acid being color-coded. Growing AV3 A. vinelandii in the presence of non-isotopically enriched sucrose and

13

C bicarbonate resulted in unlabeled PHB, as expected (SI Figure

S8). This work started with an investigation of the growth of a wildtype cyanobacteria and diazotroph. However, our investigation into the syntrophy of this coculture ultimately led us to develop a system where both carbon and nitrogen are shared and growth occurs in the absence of either fixed carbon or nitrogen. The engineered cocultures serve both to support our model of cross-feeding and represent an interesting synthetic biology platform (Figure 1). This system could be used to make a wide variety of interesting compounds/polymers in addition to PHB either with the system we describe or with additionally engineered A. vinelandii and/or S. elongatus strains. Engineered strains of



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Azotobacter vinelandii that overproduce either polyhydroxybutyrate or alginate have already been described,29 and it seems likely that similar approaches would be amenable to ∆nifL A. vinelandii. Work towards this goal is currently underway in our lab. The growth dependency of the coculture on the starting cell ratio and starting cell number of cscB S. elongatus cells suggests the need for investigating coculture growth strategies that move beyond batch systems and possibly the development of a faster growing cscB cyanobacteria coculture partner, such as S. elongatus UTEX 2973.21 This coculture could serve as an interesting system for more basic science applications as well. Specifically, we envision that this system could be a useful model system for studying obligate mutualisms since both organisms are well-studied and their genomes have been sequenced.8,30 In this work, we design a system where both fixed carbon and fixed nitrogen are shared, allowing the coculture to grow from air, water, phosphate and trace metals. Designed microbial communities are underutilized in biotechnology applications and we hope that this research will join a growing body of work that suggests the designed coculture of microorganisms can lead to more sustainable materials for the chemical industry. METHODS Cell culture. Azotobacter vinelandii was cultured in modified Burke’s medium.31 Synechococcus elongatus was cultured in BG-11 medium32 with 25 µmoles photons m-2 s-1 irradiance of cool white fluorescent light from a 9 W GE Spiral bulb. The Azotobacter vinelandii and Synechoccocus elongatus cocultures were cultured in SAV media at 22 °C under constant 25 µmoles photons m-2 s-1 irradiance with shaking at 150 rpm. In order to


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make SAV media the following solutions should be prepared: solution 1 is composed of 0.2 g/L KH2PO4 and 0.8 g/L K2HPO4; solution 2 is prepared by adding 0.09 g CaCl2*2H2O, 0.2 g MgSO4*7H2O and 0.005 g FeSO4*7H2O to 1 L of DI water; 1000X Burke’s micronutrient solution contains 2.8 g/L H3BO3, 1.592 g/L MnSO4*H2O, 0.752 g/L Na2MoO4*2H2O, 0.24 g/L ZnSO4*7H2O, 0.04 g/L of NiCl*6H2O and CuSO4*5H2O and 0.056 g/L CoSO4*7H2O. 100X BG-11 micronutrient solution contains 0.3 g/L H3BO3, 0.2 g/L MnCl2*4H2O, 0.048 g/L Na2MoO4*2H2O, 0.023 g/L ZnSO4*7H2O, 0.01 g/L CuSO4*5H2O, 0.028 g/L NaVO3 and 0.011 g/L of CoSO4*7H2O. SAV media preparation: to make a 1 L solution, fill a beaker with 700 mL of DI water. From BG-11 media31 add 5 mL of 15 g/L MgSO4, 4 mL of 9 g/L CaCl2 stock, 4 mL of 1.5 g/L citric acid stock, 5 mL of 0.2 g/L EDTA stock, 4 mL of a 5 g/L Na2CO3 stock and 10 mL of a 100X BG-11 micronutrients stock.31 From Burke’s media add 50 mL of 20X solution 1, 200 mL of 5X solution 2 and 1 mL of 1000X Burke’s micronutrients solution. Then add 0.338 g of monosodium glutamate for a final concentration of 2 mM. Fill to 1 L. Autoclave the solution and let cool. Then, add 12 mL of sterile 0.5 mg/mL iron (II) citrate to the SAV media. CAV media follows the same recipe except EDTA, citric acid and glutamate were eliminated. Also, iron (II) sulfate was added instead of iron (II) citrate keeping the moles of iron added the same. 150 mM of NaCl was added to the media and the pH of CAV media was adjusted to 8.4 with KOH. Also, just prior to cell culture for every 25 mL CAV media, 0.05 g of sodium bicarbonate was added as a sterile 10% solution in water to speed growth. The addition of sodium bicarbonate is not necessary for growth. A. vinelandii and S. elongatus were grown to mid log phase growth over 2 and 4



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d, respectively, and 150 uL of washed A. vinelandii and 300 uL of washed S. elongatus was added to 25 mL of SAV or CAV media (These amounts varied when investigating cell growth as a function of cell ratio). The CAV cultures were grown in batch at 30 °C under 25 µmoles photons m-2 s-1 irradiance of cool white fluorescent light without shaking. The A. vinelandii and S. elongatus cells were washed thoroughly with SAV or CAV media to remove any excess sucrose or nitrate. Optical densities of non-shaking cultures were determined after vortexing the solutions so as to homogenize them. Each optical density reading was its own experiment. Strain construction. All cloning was done using Golden Gate cloning.33 Sucrose permease (cscB) from E. coli (ATCC 700927) was cloned into neutral site 2 under either an IPTG-inducible promoter or a psbA1 promoter. Exact sequences inserted are found in the supporting information. A kanamycin resistance cassette was used for selection purposes. Dialysis experiments. Dialysis experiments were performed with dialysis sacks from Sigma-Aldrich with a MWCO of 12,000 Da (Product #: D6066-25EA). For the SAV coculture, 10 mL of sterile phosphate buffer (Solution 1, Burke’s modified media) was added to an Erlenmeyer flask (to prevent growth to high ODs in the dialysis bags) and for the CAV coculture 10 mL of sterile CAV media was added to the Erlenmeyer flask. Then, a dialysis bag was filled with 10 mLs of either S. elongatus or A. vinelandii in SAV (or CAV) media. Then an additional dialysis bag was filled with either the other coculture partner or 10 mL of sterile SAV media. The total volume of liquid in the flask was always 30 mL (10 mL of Solution 1/CAV media, 20 mL of SAV/CAV media in the dialysis bags) and there were always two sealed dialysis bags per flask (Figure 2A).


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Dialysis bags were sterilized by soaking in 100 mL of 70% ethanol for 2 hours. Before use they were washed with sterile water 3x with 100 mL for 10 min each. Chlorophyll analysis. Chlorophyll analysis was performed by extracting the chlorophyll with a 1 mM sodium dithionite 85% methanol solution and the concentration was determined based on previous work.34 13

C bicarbonate experiments and PHB isolation. To 40 mL of CAV media

containing the coculture, 0.1 g of sodium bicarbonate (labeled or unlabeled) was added and the culture was grown without shaking in a 250 mL flask under the previously described light conditions. Then, after 2 d, the coculture was transferred to a sealed flask, the headspace was flushed with nitrogen gas for 1 min, and 0.1 g of sodium bicarbonate were added to the flask again. After 3 d, the PHB was isolated and hydrolyzed to crotonic acid via a published method.35 The concentration of PHB was determined via a published

UV-Vis

method.35

Crotonic

acid

was

derivatized

with

N,O-

bis(trimethylsilyl)acetamide (BSA) from Sigma Aldrich (BSA Derivatization Grade 33036). For GC-MS analysis, the crotonic acid was extracted from aqueous sulfuric acid into dichloromethane. For every mole of crotonic acid in the DCM, 2 moles of BSA were added. This mixture was allowed to react for 10 min at room temperature and then injected onto a GC-MS. The PHB standard was obtained from Sigma Aldrich and the product number is 363502. ASSOCIATED CONTENT Supporting information. Experimental details and supporting figures. This material is available free of charge via the Internet. Additional information supporting our main data and the sequences used for golden gate cloning are available. AUTHOR INFORMATION



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Corresponding Author [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by an NSF SAGE IGERT fellowship, the Synthetic Biology Institute at UC Berkeley and from the Berkeley Chemical Biology Graduate Program (NRSA Training Grant 1 T32 GMO66698). We also would like to acknowledge Professor David Savage for providing us with S. elongatus transformation vectors and for helpful discussion. Additionally, we would like to acknowledge Professor Leonardo Curatti for generously providing the ∆nifL A. vinelandii strain. REFERENCES 1. Bader, J., Mast-Gerlach, E., Popovic, M., Baipai, R., and Stahl, U. (2010) Relevance of microbial coculture fermentations in biotechnology. J. Appl. Microbiol. 109, 371–387. 2. Goers, L., Freemont, P., and Polizzi, K. M. (2014) Co-culture systems and technologies: taking synthetic biology to the next level. J. R. Soc. Interface 11, 20140065. 3. Ducat, D. C., Avelar-Rivas, J. A., Way, J. C., and Silver, P. A. (2012) Rerouting Carbon Flux to Enhance Photosynthetic Productivity. Appl. Environ. Microbiol. 78, 2660-2668. 4. Ortiz-Marquez, J. C., Nascimento, M. D., Dublan, M., and Curatti, L. (2012) Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae. Appl. Environ. Microbiol. 78, 2345-2352. 5. Merino-Puerto, V., Schwarz, H., Maldener, I., Mariscal, V., Mullineaux, C., Herrero, A., and Flores, E. (2011) FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium Anabaena sp. Mol. Microbiol. 82, 87–98. 6. Greenwell, H. C., Laurens, L. M., Shields, R. J., Lovitt, R. W., and Flynn, K. J. (2010) Placing microalgae on the biofuels priority list: a review of the technological challenges. J. R. Soc. Interface 7, 703-726. 7. Twite, A., Hsiao, S., Onoe, H., Mathies, R., and Francis, M. (2012) Direct attachment of microbial organisms to material surfaces through sequence-specific DNA hybridization. Adv. Matter 24, 2380–2385. 8. Setubal, J. C. et al. (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J. Bacteriol. 191, 4534–4545.


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9. Clementi, F. (1997) Alginate production by Azotobacter vinelandii. Crit. Rev. Biotechnol. 17, 327–361. 10. El-Shanshoury, A. E. et al. (2013) Optimization of Polyhydroxybutyrate (PHB) production by Azotobacter vinelandii using experimental design. Int. J. Curr. Microbiol. App. Sci. 2, 227-241. 11. Revillas, J. J., Rodelas, B., Pozo, C., Martinez-Toledo, M. V., and Lopez, J. G. (2005) Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions. Amino Acids 28, 421-425. 12. Taller, B. J., and Wong, T. Y. (1989) Cytokinins in Azotobacter vinelandii culture medium. Appl. Environ. Microbiol. 55, 266-267. 13. Azcon, R., and Barea, J. M. (1975) Synthesis of auxins, gibberellins and cytokinins by Azotobacter vinelandii and Azotobacter beijerinckii related to effects produced on tomato plants. Plant and Soil 43, 609-619. 14. Galindo, E., Pena, C., Nunez, C., Segura, D., and Espin, G. (2007) Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fact. 6, 1475-2859. 15. Page, W. J., and Knosp, O. (1989) Hyperproduction of Poly-beta-Hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl. Environ. Microbiol. 55, 1334-1339. 16. Bali, A. et al. (1992) Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl. Environ. Microbiol. 58, 1711-1718. 17. Khosravi-Darani, K. et al. (2013) Microbial production of poly(hydroxybutyrate) from C1 carbon sources. Appl. Microbiol. Biotechnol. 97, 1407-1424. 18. Nath, A., Dixit, M., Bandiya, A., Chavda, S., and Desai, A. J. (2008) Enhanced PHB production and scale up studies using cheese whey in fed batch culture of Methylobacterium sp. ZP24. Bioresource Technology. 99, 5749-5755. 19. Golden, S. S., Brusslan, J., and Haselkorn, R. (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 153, 215-231. 20. Johnsborg, O., Eldholm, V., and Havarstein, L. S. (2007) Natural genetic transformation: prevalence, mechanisms and function. Res. Microbiol. 158, 767–778. 21. Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., Koppenaal, D. W., Brand, J. J., and Pakrasi, H. B. (2015) Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Scientific Reports 5, DOI: 10.1038/srep08132. 22. Tsinoremas, N. F., Kutach, A. K., Strayer, C. A. and Golden, S. S. (1994) Efficient gene transfer in Synechococcus sp. strains PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination. J. Bacteriol. 176, 6764–6768. 23. Ditty, J. L., Canales, S. R., Anderson, B. E., Williams, S. B., and Golden, S. S. (2005) Stability of the Synechococcus elongatus PCC 7942 circadian clock under directed anti-phase expression of the kai genes. Microbiology 151, 2605-2613. 24. Niederholtmeyer, H., Wolfstadter, B. T., Savage, D. F., Silver, P. A., and Way, J. C.



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(2010) Engineering cyanobacteria to synthesize and export hydrophilic products. Appl. Environ. Microbiol. 76, 3462–3466. 25. Lea-Smith, D. J., Biller, S. J., Davey, M. P., Cotton, C. A. R., Perez Sepulveda, B. M., Turchyn, A. V., Scanlan, D. J., Smith, A. G., Chisholm, S. W., and Howe, C. J. (2015) Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle. Proc. Natl. Acad. Sci. USA 101, 314-319. 26. Knoop, H., Grundel, M., Zilliges, Y., Lehmann, R., Hoffmann, S., Lockau, W., and Steuer, R. (2013) Flux balance analysis of cyanobacterial metabolism: The metabolic network of Synechocystis sp. PCC 6803. PLoS Comput. Biol. 9, e1003081. 27. Kurz, W. G., and LaRue, T. A. (1972) Metabolism of glycolic acid by Azotobacter chroococcum PRL H62. J. Microbiol. 19, 321-324. 28. Mulo, P., Sicora, C., and Aro, E. M. (2009) Cyanobacterial psbA gene family: optimzation of oxygenic photosynthesis. Cell Mol. Life Sci. 66, 3697-3710. 29. Segura, D., Guzman, J., and Espin, G. (2003) Azotobacter vinelandii mutants that overproduce poly-beta-hydroxbutyrate or alginate. Appl. Microbiol. Biotechnol. 63, 159-163. 30. Chen, Y., Holtman, C. K., Magnuson, R. D., Youderian, P. A., and Golden, S. S. (2008) The complete sequence and functional analysis of pANL the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942. Plasmid 59, 176–192. 31. D’Mello, R., Hill, S., and Poole, R. K. (1994) Determination of the oxygen affinities of terminal oxidases in Azotobacter vinelandii using the deoxygenation of oxyleghaemoglobin and oxymyoglobin: cytochrome bd is a low-affinity oxidase. Microbiology 140, 1395–1402. 32. Stanier, R., Kunisawa, R., Mandel, M., and Cohen-Bazire, G. (1971) Purification and properties of unicellular blue-green algae (order Cchroococcales). Bacteriol. Rev. 35, 171–205. 33. Engler, C., Kandzia, R., and Marillonnet, S. (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3, e3647. 34. Porra, R. J. (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth. Res. 73, 149-156. 35. Law, J. H., and Slepecky, R. A. (1961) Assay of poly-β-hydroxybutyric acid. J. Bacteriol. 82, 33-36.

Figure 1. Proposed models of syntrophy. (A) A. vinelandii, but not S. elongatus can metabolize glutamate. S. elongatus receives nitrogen, in the form of ammonia, from A. vinelandii. (B) cscB S. elongatus supplies A. vinelandii with a reduced carbon source that enables the coculture to grow in the absence of glutamate. (C) cscB S. elongatus and AV3


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A. vinelandii grow to higher optical densities and synthesize more chlorophyll suggesting ammonia transport was a bottleneck in the metabolic cross-feeding model.

Figure 2. Growth assays of the coculture and the corresponding monocultures grown in SAV media. (A) The S. elongatus-A. vinelandii coculture has enhanced growth over the monocultures. S. elongatus does not grow in the absence of A. vinelandii in SAV media. (B) The growth of the coculture in dialysis bags is depicted. In the leftmost flask is one dialysis bag with S. elongatus (green) and one dialysis bag with A. vinelandii. In the rightmost flask is one dialysis bag with S. elongatus (now colorless) and one with SAV media. S. elongatus benefits from the presence of A. vinelandii in terms of (C) OD and (D) chlorophyll production. A. vinelandii does not benefit from the presence of S. elongatus. Each timepoint was performed in biological triplicate. (+/- SD, N=3)

Figure 3. Engineered and wildtype cocultures grown without any fixed organic carbon or nitrogen. (A) The wildtype coculture does not grow over a period of 8 d and (B) exhibits less chlorophyll production than the engineered cocultures. The cscB S. elongatus + wt A. vinelandii coculture grows slower and synthesizes less chlorophyll than the cscB S. elongatus + AV3 A. vinelandii combination over 8 d. The starting cell ratios of the wt+wt coculture=96:4 S. elongatus:A. vinelandii, wt+AV3=96:4, cscB+wt=95:5, cscB+AV3=96:4. (C) Dialysis experiments were performed in CAV media. OD was measured after 10 d in solution (+/- SD, N=3).

Figure 4. Coculture growth as a function of cell ratio and density. (A) The cell ratios after 24 d in CAV media, diluting 1:10 every 10 d, were determined by flow cytometry. A. vinelandii cells were the viable non-autofluorescent cells. S. elongatus were the viable autofluorescent cells. There was a larger relative amount of S. elongatus cells in the cscB+AV3 coculture. Viability was determined via CFDA dye (+/- SD, N=3). (B and C) The starting cell ratio affected the cell growth of the cscB+AV3 coculture at various starting cell densities. Ratios were reported as a percentage of S. elongatus cells in initial cells used to inoculate the media, so 62% S. elongatus indicates a ratio of 62:38 S. elongatus:A. vinelandii. (D) The starting cell density affects the cell growth of the cscB+AV3 coculture (+/- SD, N=3). Figure 5. 13C bicarbonate labeling experiment. PHB either from a standard or that was extracted from the cscB-AV3 coculture that was grown in the presence of 13C bicarbonate was hydrolyzed to crotonic acid, derivatized with BSA, and analyzed by GC-MS. The resulting isotopic patterns were compared. The labeling experiment demonstrates that the majority of the PHB monomer contains four 13C incorporations (peak at 147 m/z).



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Figure 1 81x134mm (300 x 300 DPI)


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Figure 2 91x120mm (300 x 300 DPI)



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For Table of Contents Use Only 74x33mm (300 x 300 DPI)


S. elongatus Coculture for Chemical Photopr - ACS Publications

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