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ScienceDirect Engineering cyanobacteria for direct biofuel production from CO2 Philipp Savakis1 and Klaas J Hellingwerf1,2 For a sustainable future of our society it is essential to close the global carbon cycle. Oxidised forms of carbon, in particular CO2, can be used to synthesise energy-rich organic molecules. Engineered cyanobacteria have attracted attention as catalysts for the direct conversion of CO2 into reduced fuel compounds.Proof of principle for this approach has been provided for a vast range of commodity chemicals, mostly energy carriers, such as short chain and medium chain alcohols. More recently, research has focused on the photosynthetic production of compounds with higher added value, most notably terpenoids. Below we review the recent developments that have improved the state-of-the-art of this approach and speculate on future developments. Addresses 1 Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands 2 Photanol BV, Amsterdam, The Netherlands Corresponding author: Hellingwerf, Klaas J ([email protected])

Current Opinion in Biotechnology 2015, 33:8–14 This review comes from a themed issue on Energy biotechnology Edited by E Terry Papoutsakis and Jack T Pronk

http://dx.doi.org/10.1016/j.copbio.2014.09.007

factories’ for these organisms/systems (see also Figure 1). At the laboratory-scale biosolar cell factories have now proven to be functional for sucrose, ethanol,   L-lactic acid and 2,3-butanediol [4,5 ,6 ,7]. For a limited range of compounds pilot-scale facilities have been set up and are currently being tested for economic competitiveness. To a significant extent the rapid expansion of the range of products that can be made by cyanobacteria, engineered to carry out ‘photofermentative’ metabolism, was facilitated by a spill-over of knowledge from conventional fermentation approaches, in particular from Escherichia coli and Saccharomyces cerevisiae, which have been engineered to produce a wide range of products. In the cyanobacterial field, most metabolic engineering studies have been carried out using the two-model organisms Synechocystis sp. PCC 6803 (Synechocystis 6803) and Synechococcus elongatus sp. PCC 7492. Synechococcus sp. PCC 7002 (Synechococcus 7002) and Anabaena sp. PCC 7120 (Anabaena 7120), however, are receiving increased attention. A similar approach of ‘direct conversion’ is also possible for eukaryotic oxygenic phototrophs, like green algae or brown algae. However, the generally much more complex genetic engineering required for these organisms has hampered rapid progress so far [8].

0958-1669/# 2014 Elsevier Ltd. All rights reserved.

Biofuels and other products of engineered cyanobacteria

Introduction: ‘light-driven conversion’ Cyanobacteria are photosynthetic prokaryotes that can use photon energy to ultimately transfer electrons from water to carbon dioxide, generating more reduced molecules in the process. The introduction of heterologous, mostly catabolic, pathways into the metabolism of cyanobacteria allows production of a wide range of fuel and commodity products from CO2, light and water [1,2]. In the recent past this approach has matured so that by now for a large range of compounds proof of principle has been provided (for review see: [3]; Angermayr, Thesis, 2014). Quantitative evaluation of these production systems leads to the conclusion that in many cases the majority (i.e. >50%) of the CO2 fixed by the engineered cyanobacterium is directly converted into product [4]. Angermayr et al. introduced the term ‘biosolar cell Current Opinion in Biotechnology 2015, 33:8–14

With fuel applications in mind, there are several interesting classes of molecules available. Figure 2 gives an overview of heterologous pathways that have been introduced into cyanobacteria and the corresponding product titres that were achieved. Hydrogen, although versatile in its applications, will not be discussed in this review. Short chain alcohols can be used as drop-in automotive fuels. The highest titres for a photosynthetically produced biofuel have been reported by Gao et al. for ethanol (5.5 g/L after 26 days) [6]. Cyanobacterial production of acetone [9], isopropanol [10] and 1,2-propanediol [11] was reported recently. Lan and Liao increased butanol titres [12,13]. 2,3-Butanediol synthesis was achieved in Synechococcus 7942 and Synechocystis 6803 [7,14,15]. Shen and Liao reported production of 2-methyl-1-butanol in Synechococcus 7942 [16]. Because of their high energy content, fatty acids and their alcohol derivatives and alkane derivatives are attractive www.sciencedirect.com

Engineering cyanobacteria for biofuel production Savakis and Hellingwerf 9

Figure 1

hν

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Schematic representation of a cyanobacterial cell factory. In the thylakoids, photon energy drives water splitting, reduction of NADP and phosphorylation of ADP. Carbon dioxide gets assimilated in the CalvinBenson-Bassham cycle at the cost of NADPH and ATP. Fixed carbon can then either be channelled into biomass or into a production pathway.

fuels. Following the work on Synechocystis 6803 [17], photosynthetic production and excretion of free fatty acids (FFAs), through overexpression of a thioesterase, were recently also achieved in Synechococcus 7942 [18] and in Synechococcus 7002 [19]. Using an alternative approach, that is, overexpression of the endogenous acyl-ACP reductase, Kaiser et al. demonstrated excretion of FFAs in Synechococcus 7942 [20]. In the same study, production of triacylglycerols and wax esters was reported. Overproduction of alkane biosynthesis genes [21] from various cyanobacteria in Synechocystis 6803 led to an increase of heptadecane and heptadecene content [22]. Mutants harbouring NADPH-dependent fatty acyl-CoA reductase showed increased levels of C15–C17 fatty alcohols [23]. Terpenoids are an extremely diverse class of molecules, highly interesting for a wide variety of fuel-chemical, bulk-chemical, and fine-chemical applications. Production of isoprene in cyanobacteria was first reported by Lindberg et al. [24]. Recently, also production of bphellandrene was demonstrated [25,26]. Limonene production has been reported in Anabaena 7120 [27], Synechocystis 6803 [28] and Synechococcus 7002 [29]. Sesquiterpenes are attractive jet fuels or precursors thereof. Photosynthetic production of the sesquiterpene a-bisabolene was demonstrated in Synechococcus 7002; inactivation of glycogen synthesis in this strain did not positively influence terpenoid production [29]. Englund et al. showed that deletion of squalene-hopene cyclase in www.sciencedirect.com

Synechocystis 6803 leads to a mutant that accumulates squalene [30]. The terpenoid precursors dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) can be synthesised via the methylerythritol phosphate (MEP) or via the mevalonate pathway. Cyanobacteria utilise the former. Overproduction of MEP pathway enzymes led to increased carotenoid levels [31] and increased limonene yield [27,28]. Cells expressing the heterologous mevalonate pathway in combination with an isoprene synthase showed increased isoprene titres [32]. Thermal recycling of fossil-derived plastics contributes to CO2 emission. Conversion of the CO2 produced as the direct result of thermal recycling of polymers into new monomers would contribute to closing the carbon cycle. For the monomers ethylene [33] and Llactate [5,34,35] increased yields were reported. Proof of principle was provided for the production of D-lactate [36] and 3-hydroxybutyrate [37].

General challenges for the design of production systems A compound of interest can be produced through the introduction of a reaction that converts an endogenous metabolite. If the compound of interest is an endogenous metabolite, then its intracellular concentrations can be increased by removal of a consuming reaction. Many compounds are excreted from and/or leak out of the cytoplasm into the culture medium. For cyanobacteria, currently little is known about the underlying processes. It was shown that exporter proteins can lead to extracellular accumulation of products that otherwise would not be able to diffuse through the membrane [38]. In selected examples, overall product formation was stimulated through such proteins [4]. Fermentative pathways are attractive for biofuel synthesis as many lead to the formation of reduced compounds with high heat of combustion. In the native host, these pathways are typically operating under conditions with limited oxygen supply and can therefore include oxygen sensitive conversions, which may conflict with oxygenic photosynthesis. Mutually incompatible processes can be either changed or separated. Thus, replacing oxygen-sensitive CoA-acylating butyraldehyde dehydrogenase with oxygen-tolerant CoA-acylating propionaldehyde dehydrogenase increased butanol titres [13]. Separation can either be spatial [39] or temporal. Spatial separation can be achieved in single cells through protein complexes or specialised micro-compartments, in multicellular strains through specialised cells, and in cocultures [40,41] through different organisms. In filamentous diazotrophic cyanobacteria, vegetative cells carry out oxygenic photosynthesis, while oxygen-sensitive nitrogen fixation takes place in dedicated heterocysts. Harnessing the power of the latter, Ihara et al. targeted oxygen-sensitive formate Current Opinion in Biotechnology 2015, 33:8–14

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Figure 2

(a)

Isoprene DXP

CO2 Mannitol

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Engineering cyanobacteria for biofuel production Savakis and Hellingwerf 11

dehydrogenase to heterocysts of Anabaena 7120, which resulted in the direct reduction of CO2 to formate [42]. Temporal separation of oxygenic photosynthesis and nitrogen fixation is realised in unicellular diazotrophs [43]. These strains provide potentially interesting targets for genetic engineering. Storage compounds allow temporal separation of photon energy harvesting and utilisation. During nitrogen starvation, cyanobacteria can accumulate up to 41% of dry cell weight as glycogen [44]. Synechocystis 6803 can accumulate up to 9.5% and 11% of its dry cell weight as polyhydroxybutyrate (PHB) under nitrogen starvation and phosphorus starvation, respectively [45]. Inactivation of storage pathways should therefore allow a greater fraction of the fixed carbon to be directed into product. Through this approach, partitioning to product can only be increased in conditions under which storage pathways would be active in the wild-type organism. Inactivation of the glycogen synthesis pathway has shown success under nitrogen deplete conditions [46,47]. Inactivation of PHB biosynthesis increased yields of 3-hydroxybutyrate [37] and led to a 70% increase in heptadecane and heptadecene content, compared to the strain that still produced PHB [22]. For lactate production, no positive effect of this mutation could be observed [47]. While for an ethanol producing Synechocystis strain, inactivation of PHB synthesis did not lead to a significant increase in productivity, a PHB deficient mutant harbouring an additional copy of the ethanol production cassette showed a twofold increase in enzyme activity and, intriguingly, a threefold increase in ethanol titre [6]. This illustrates that in optimising productivity, actual limitations should be identified. To this end, a sensitivity analysis [48] was recently carried out for lactate producing Synechocystis strains [34]. At very high expression levels it could be shown that control was shifted away from the lactate dehydrogenase into other parts of the product-forming pathway, as expression of pyruvate kinase only stimulated product formation in this latter strain [5]. An alternative to the removal of competing pathways, is the manipulation of driving forces. In cyanobacteria decarboxylation reactions [7,15,16,49–51] and cleavage of phosphoester bonds [52] have been used for this.

Coupling of an uphill reaction to ATP-hydrolysis has been successful [12]. NADPH can be directly recycled by the thylakoids and is presumably more abundant than NADH. NADH-dependent reactions can therefore be driven through expression of a transhydrogenase [11,15,35,38] or by replacement with enzymes that utilise NADPH [6,11,15]. In the pursuit of ever-higher titres, product toxicity can limit yield. A number of studies have investigated transcriptional responses induced by added or produced solvent [53–57]. Anfelt et al. identified, among other proteins, HspA to increase tolerance of Synechocystis towards butanol. Production of free (particularly unsaturated) fatty acids in Synechococcus 7492 seems to be limited by negative physiological effects [18], most probably product intercalation in cellular and thylakoid membranes [58]. For volatile products continuous extraction can be used for sustained production [28,29,49]. This alleviates not only product toxicity, but also potential product (feedback) inhibition. Although eventual large-scale production will most probably occur under a day-night regime, most laboratory studies have investigated productivity under continuous light. For a lactate producing strain, productivity under light-dark conditions did not significantly differ from production under constant light [35]. The industrial application of processes developed on the laboratory scale requires upscaling. Details of designing photobiorectors for these purposes are beyond the scope of this review. For this the reader is referred to [59]. Upscaling involves growing cyanobacteria in large quantities. The more cell doublings are required to achieve the desired amount of biomass, the greater the probability that a spontaneous mutation that increases growth rate will be selected for. For production systems this means that any mutant that diverts fixed carbon away from a production pathway will be enriched (See [60] for an overview on genetic instability in cyanobacteria). Therefore it may be of importance to separate the growth phase and the production phase, so that mutations leading to decreased productivity will not be positively selected for. To this end, production pathways can be placed under

( Figure 2 Legend Continued ) (a) Intermediary metabolism of a typical cyanobacterium under photoautotrophic growth conditions, including heterologous, biofuel-forming pathways. Fuels are coloured according to the metabolite they originate from. Major carbon sinks are underlined. Abbreviations: 2 PG: 2-phosphoglycerate, 3 PG: 3-phosphoglycerate, ACP: acyl carrier protein, CoA: coenzyme A, DHAP: dihydroxyacetone phosphate, DMAPP: dimethylallylpyrophosphate, DXP: 1-deoxyxylulose-5-phosphate, E4P: erythrose-4-phosphate, F1,6BP: fructose-1,6bisphosphate, F6P: fructose-6-phosphate, G1P: glucose-1-phosphate, G6P: glucose-6-phosphate, GABA: g-hydroxybutyrate, GAP: glyceraldehyde3-phosphate, GPP: geranyl pyrophosphate, HMBPP: 4-hydroxy-3-methylbut-2-enyl diphosphate, HMG-CoA: hydroxymethylglutaryl CoA, IPP: isopentenyl pyrophosphate, MEP: 2-methylerythritol-4-phosphate, OA: oxaloacetate, OG: oxoglutarate, PEP: phosphoenolpyruvate, Ru1,5BP: ribulose-1,5-bisphosphate, Ru-5-P: ribulose-5-phosphate, R-5-P: ribose-5-phosphate, S1,7BP: sedoheptulose-1,7-bisphosphate, S7P: sedoheptulose-7-phosphate, SSA: succinic semialdehyde, Xu-5-P: xylulose-5-phosphate. (b) Production overview. Maximum titres (in the respective study) of excreted products are shown at the time points indicated. The time axis does not distinguish between constitutive systems and inducible systems, in which concentrations are typically measured x days after induction. Colours used reflect the cyanobacterial native metabolite that the product originates from. www.sciencedirect.com

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tight control. Huang and Lindblad developed a tightly regulated promoter system [61] but the fact that it is induced by anhydrotetracyclin might forestall large scale applications. Ideally, inexpensive inducers, such as light, would be used. Thus, Miyake et al. developed a greenlight inducible lysis system, based on the cpcG2 promoter of Synechocystis 6803 [62]. Advances in the genetic manipulation of cyanobacteria were reviewed recently [63]. Increasing complexity of heterologous pathways, and the need to manipulate host metabolism have led to the development and improvement of a number of counterselection systems based on B. subtilis sacB [17,51,64–66], E. coli mazF [67], and organic acid sensitivity conferred by AcsA [68]. Metabolic engineering of heterotrophs has benefitted considerably from metabolic models. For cyanobacteria, various genome scale models have been constructed [69– 71]. Although highly desirable, the construction of dynamic models is thus far limited by the availability of kinetic data [72]. Instead, flux balance analysis (FBA) is used. FBA predicts a flux distribution in which an objective function is optimised (e.g. maximisation of biomass formation) [73]. In a system, where growth does not depend on product formation (such as in a photosynthetic cell factory), biomass formation as the objective function would lead to a flux distribution that does not allow for product formation, while optimisation for product formation would not predict growth. Therefore, current models of production strains use product formation as objective function, while growth is fixed to a certain value [74,75]. Such a priori constraints limit the predictive value of these models, reducing their applicability considerably.

[17,18,19,20,58,76] and fatty alcohols [23] producing strains. Furthermore, Synechococcus 7942 engineered to produce 2-methyl-1-butanol accumulated 1-propanol and isobutanol as by-products [16]. This approach, however, shows distinct disadvantages for the production of commodity chemicals. At present, the cost of the most developed biofuel, plantderived biodiesel, is still several times that of fossil fuels. As cyanobacteria do not need to devote fixed carbon to the synthesis of trunks, roots etc., production yields for a direct conversion approach per m2 could be significantly higher. With additional costs for downstream processing and for operation of photobioreactors and, most significantly for maintaining axenic conditions, the price for solar biofuels is still considerably higher than for the fossil competitor product. This is true in particular when no cost is placed on carbon emission. Therefore, for a development towards a more mature technology based on ‘direct conversion’ it may be wise to initially concentrate on the production of higher-value-added compounds. Future developments in strain selection and engineering, bioreactor design and processing technology then may pave the way for the production of fuels, using the direct conversion approach that can economically compete with their fossil counterparts.

Acknowledgements The authors thank Filipe Branco dos Santos and Pascal van Alphen for helpful discussions and S. Andreas Angermayr for critical review of the manuscript. This review was written within the research programme of BioSolar Cells, co-financed by the Dutch Ministry of Economic Affairs.

References and recommended reading The ideal cyanobacterial host The ideal production host is hard to specify. Rather, different desired properties can be formulated, depending on the nature of the process. Generally, genetic manipulation should be straightforward and engineered strains should show a high degree of stability. Growth should be fast and robust, as well as photosynthetic efficiency and carbon fixation rate could be high. To use off-gas, growth at elevated CO2 concentrations should be possible. Growth in seawater would reduce competition with the freshwater use for food purposes. Product excretion and flocculation can facilitate harvesting. Cultures should show resistance to product consuming contaminants and grazers.

Scientific and economic prospects Commercial fuels usually are mixtures of compounds. Similarly, production of biofuel mixtures by photosynthetic microbes could be advantageous; even more so as many enzymes show inherent promiscuity which may lead to product diversification. Mixtures with different chain lengths were reported for alkanes [22], fatty acids Current Opinion in Biotechnology 2015, 33:8–14

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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Angermayr SA, van der Woude AD, Correddu D, Vreugdenhil A, Verrone V, Hellingwerf KJ: Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803. Biotechnol Biofuels 2014, 7:99. Conclusive demonstration that increasing pyruvate concentrations is only beneficial when the production pathway does not have full control on the rate of product formation.

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Gao Z, Zhao H, Li Z, Tan X, Lu X: Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria. Energy Environ Sci 2012, 5:9857-9865. www.sciencedirect.com

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The highest yield for a biofuel so far obtained through the process of ‘direct conversion’. 7.

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10. Kusakabe T, Tatsuke T, Tsuruno K, Hirokawa Y, Atsumi S, Liao JC, Hanai T: Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light. Metab Eng 2013, 20:101-108. 11. Li H, Liao JC: Engineering a cyanobacterium as the catalyst for the photosynthetic conversion of CO2 to 1,2-propanediol. Microb Cell Factories 2013, 12:4. 12. Lan EI, Liao JC: ATP drives direct photosynthetic production of  1-butanol in cyanobacteria. Proc Natl Acad Sci U S A 2012, 109:6018-6023. The authors describe how the use a thermodynamically unfeasible reaction can be circumvented by coupling an alternative reaction to ATP hydrolysis. 13. Lan EI, Ro SY, Liao JC: Oxygen-tolerant coenzyme A-acylating  aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria. Energy Environ Sci 2013, 6:2672-2681. Further pathway optimisation dramatically improved butanol yield. 14. Oliver JWK, Machado IMP, Yoneda H, Atsumi S: Combinatorial optimization of cyanobacterial 2,3-butanediol production.  Metab Eng 2014, 22:76-82. Optimisation of meso-butanediol production to a very high degree of carbon partitioning. It is shown that a general recipe for optimisation of regulatory elements cannot be provided. Rather, every operon needs to be optimised individually. 15. Savakis PE, Angermayr SA, Hellingwerf KJ: Synthesis of 2,3butanediol by Synechocystis sp. PCC6803 via heterologous expression of a catabolic pathway from lactic acid- and enterobacteria. Metab Eng 2013, 20:121-130. 16. Shen CR, Liao JC: Photosynthetic production of 2-methyl-1butanol from CO2 in cyanobacterium Synechococcus elongatus PCC7942 and characterization of the native acetohydroxyacid synthase. Energy Environ Sci 2012, 5:95749583.

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17. Liu X, Sheng J, Curtiss R 3rd: Fatty acid production in genetically modified cyanobacteria. Proc Natl Acad Sci U S A 2011, 108:6899-6904.

34. Angermayr SA, Hellingwerf KJ: On the use of metabolic control  analysis in the optimization of cyanobacterial biosolar cell factories. J Phys Chem B 2013, 117:11169-11175. Application of metabolic control analysis to product formation in cyanobacteria.

18. Ruffing AM, Jones HDT: Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942. Biotechnol Bioeng 2012, 109:2190-2199.

35. Angermayr SA, Paszota M, Hellingwerf KJ: Engineering a cyanobacterial cell factory for production of lactic acid. Appl Environ Microbiol 2012, 78:7098-7106.

19. Ruffing AM: Improved free fatty acid production in cyanobacteria with Synechococcus sp. PCC 7002 as host. Synth Biol 2014, 2:17.

36. Varman AM, Yu Y, You L, Tang YJ: Photoautotrophic production of D-lactic acid in an engineered cyanobacterium. Microb Cell Factories 2013, 12:117.

20. Kaiser BK, Carleton M, Hickman JW, Miller C, Lawson D, Budde M,  Warrener P, Paredes A, Mullapudi S, Navarro P et al.: Fatty aldehydes in cyanobacteria are a metabolically flexible precursor for a diversity of biofuel products. PLoS ONE 2013, 8:e58307. The authors describe excretion of free fatty acids through a novel approach (overproduction of endogenous acyl-ACP reductase), potentially allowing for photosynthetic production of fatty acids using nontransgenic strains. Furthermore, synthesis of triacylglycerol and wax esters was demonstrated.

37. Wang B, Pugh S, Nielsen DR, Zhang W, Meldrum DR: Engineering cyanobacteria for photosynthetic production of 3hydroxybutyrate directly from CO2. Metab Eng 2013, 16C:68-77.

21. Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial biosynthesis of alkanes. Science 2010, 329:559-562. 22. Wang W, Liu X, Lu X: Engineering cyanobacteria to improve photosynthetic production of alka(e)nes. Biotechnol Biofuels 2013, 6:69. www.sciencedirect.com

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