rinro

C

4, U

Reviewcyanobacteria capture solar energy and store it as chemicalenergy of their biomass (bioenergy). Thus, agriculturemight serve as a source of food and bioenergy. Bioenergyis mostly captured by photosynthetic CO2 fixation, there-fore, carbon-containing biofuels have a varied tendency tobe carbon neutral after combustion. The extent to whichthis is accomplished largely depends on the nature of thefeedstock, the agricultural practice, and the industrialprocess, and thus it might proportionally contribute toclimate change mitigation [3]. First-generation biofuelswere based on edible feedstocks. Consequently, severalalternative feedstocks have been proposed, mainly to alle-viate food-bioenergy competition and land use change,leading to second generation or more advanced biofuels [4].

The use of microbial cell factories for bioenergy orrelated purposes, although not new, has regained attentionas a result of increasing pressure for higher productivities,

This review comments on some of the most significantachievements in genetic engineering of microalgae andcyanobacteria and discusses some cases in which biochem-ical incompatibility between the recombinant pathwaysand host metabolism has prevented further progress. Asa complementary alternative, we propose development ofmultispecies microbial cell factories comprising optimizedparts as specialized metabolic modules to allow biochemi-cal compartmentalization in complex metabolic networks(Figure 1).

Progress and constraints of genetic engineering ofphotosynthetic microbial cell factoriesMicroalgae and cyanobacteria may be engineered to pro-duce a target product and there are numerous exampleswhere this has been achieved (Table 1). One of the mostremarkable accomplishments has been the direct conver-sion of CO2 into alcohols, partially bypassing the complex-ity of the formation of biomass. Recombinant ethanolproduction up to 5.5 g/l has been obtained in cyanobacteria[13]. Also, isobutyraldehyde that can be used as a precursor

0167-7799/$ see front matter

2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2013.05.006

Corresponding author: Curatti, L. (lcuratti@fiba.org.ar).Genetic engineeringmicrobial cell factoalternative for bioeJuan Cesar Federico Ortiz-Marquez1, Mauand Leonardo Curatti1

1 Instituto de Investigaciones en Biodiversidad y Biotecnologadel Plata, Buenos Aires, Argentina2Ocean Sciences, University of California, Santa Cruz, CA 9506

There is currently much interest in developing technolo-gy to use microlgae or cyanobacteria for the productionof bioenergy and biomaterials. Here, we summarizesome remarkable achievements in strains improvementby traditional genetic engineering and discuss commondrawbacks for further progress. We present generalknowledge on natural microalgalbacterial mutualisticinteractions and discuss the potential of recent devel-opments in genetic engineering of multispecies micro-bial cell factories. This synthetic biology approach wouldrely on the assembly of complex metabolic networksfrom optimized metabolic modules such as photosyn-thetic or nitrogen-fixing parts.

Photosynthetic microbes for the production ofbioenergy and biomaterialsIncreasing food and energy demand, global climate change,and general environmental decay are some of the maincurrent challenges for humans. Biofuels, among otheralternative energy sources, have great potential to harmo-nize the foodenergyenvironment trilemma [1,2]. Photo-synthetic organisms including plants, algae, and of multispecieses as anergy production

Do Nascimento1, Jonathan Philip Zehr2,

onsejo Nacional de Investigaciones Cientficas y Te cnicas. Mar

SA

novel bioproducts, and environmental protection. It iswidely appreciated that the microbial world contains byfar the greatest fraction of biodiversity in the biosphere,and thus a corresponding innovation potential [5].According to the advantages listed in Box 1, there iscurrently much interest in developing the technologyfor the use of photosynthetic microorganisms, such aseukaryotic microlgae or cyanobacteria [6,7]. Althoughsome startup companies are already attempting to com-mercialize algal fuels [8], their actual potential is still amatter of debate [9].

Identifying suitable microalgae strains is usually a start-ing point in the roadmap towards microalgae-based technol-ogy development. The most appreciated traits for the idealmicroalga are listed in Box 2 [6,7]. Currently, there are noavailable strains excelling in all these traits, which is notsurprising considering that, conversely to the developmentof modern plant crops, there have been no breeding programsfor microalgae [10]. Although natural microalgae or cyano-bacterial isolates that exceed current yields may exist, it isexpected that breeding and/or genetic engineering would berequired to achieve industrial production [7,1012] Box 3.Trends in Biotechnology, September 2013, Vol. 31, No. 9 521

Box 1. Main advantages of microalgae or cyanobacteria as

biofuels feedstock

At their exponential growth rate they can double their biomass inperiods as short as 3.5 h.

They can be produced almost year round under favorableweather.

For oleaginous microalgae, the projected oil productivity perhectare would exceed by tenfold that of the best oilseed crops.

Despite being aquatic organisms, the culture systems (openponds or photobioreactors) demand less water than terrestrialcrops, minimizing pressure on freshwater resources.

They can be cultivated in brackish water on nonarable land, notincurring land-use change and associated environmental damage,and competence with the production of food, fodder, and otherbenefits from crops.

They can take the most vital nutrients (CO2, nitrogen, phosphorus,and others) from industrial or municipal waste, helping out tomanage waste disposal.

Microalgae cultivation does not require herbicides or pesticidesapplication.

They can also produce valuable co-products such as proteins andresidual biomass after oil extraction, which may be used as feedor fertilizer, or fermented to produce ethanol or methane.

Reviewof a variety of chemicals, including isobutanol, has beenproduced with a projected productivity that would exceedby five- to sixfold estimates for corn and cellulosic ethanolproduction on a land area basis. Isobutyraldehyde-over-producing strains were further modified to produce isobu-tanol as a better substitute for gasoline than ethanol [14].

However, the success of importing a heterologous path-way is influenced by a variety of drawbacks that mayinclude combinations of different aspects.

Some strains produce biohydrogen either as a result of theirmetabolism, by means of hydrogenase and/or nitrogenaseenzymes or indirectly by biomass fermentation with appropriatemicroorganisms.Incompatibility of oxygenic photosynthesis withanaerobic metabolism1-Butanol, a likely even-better substitute of gasoline thanisobutanol, was successfully produced at up to 15 g/l in

Box 2. Most desired characteristics of the ideal

photosynthetic microbial cell factory

High efficiency of light capture and biomass yield (growth rateand final culture density).

High production of lipid or any other useful energy carrier.Production of biomass and energy carrier at the same time.

Large cells and/or flocculation properties to facilitate harvesting. Thin cell walls or with structural characteristics for easy intracel-lular products extraction.

Tolerance to high light intensity and oxygen concentration. Resistance to contamination with other microorganisms orpredators.

High efficiency of the use of cellular nitrogen and phosphorus andhigh nutrient-recycling capacity and/or ability to utilize abundantand/or inexpensive alternative sources of nitrogen, phosphorus,and other macroelements.

Amenability to genetic analysis and manipulation. Probably less important aspects such as minimizing respirationrates to minimize carbon dissimilation, decreasing the rate ofphotoacclimation to low light to slow down self-shading aftertransition to high light, lowering the carbon:nitrogen ratio of corenitrogenous components and the amount of carbon allocated tocell structure, and the potential to contribute to enhance biofuelsyield.

522recombinant Escherichia coli cells from glucose [15]. Con-versely, the more challenging task of producing 1-butanolfrom CO2 in cyanobacteria remains more elusive becauseonly up to 0.0145 g/l over 7 days could be produced and onlyunder dark, anaerobic conditions at the expense of internalcarbohydrate storage [16].

Hydrogen production is catalyzed by combinations ofoxygen-sensitive hydrogenases and/or nitrogenases. Hy-drogen production is normally low in cyanobacteria andmicroalgae and thus genetic engineering approacheshave been pursued to boost that capacity by means of

Box 3. Multispecies microbial cell factories

Microbial consortia usually perform more complex tasks thanmonocultures and can perform functions that are difficult or evenimpossible for individual strains or species.This approach may alleviate pressure towards increasing yields of

heterologous pathways in selected expression platforms, for whichoverall metabolic coupling is weak and/or mostly poorly under-stood. In turn, the concept of genetic engineering of multispeciesmicrobial cell factories might rely, as a starting point, on microbeswith one or a few outstanding properties as metabolic/productionmodules, which can be further optimized for the same or othereasier-to-accomplish tasks. This approach takes advantage of thepossibility of metabolic compartmentalization between or amongcells with different characteristics and/or onto which the burden ofoverexpressing multiple genes can be split among the partners ofthe consortium. Communication for trading extracellular metabo-lites is a key aspect of the principle which on one hand increases thelevel of complexity and on the other hand might provide opportu-nities for additional possibilities of regulation of the productiveplatforms and/or removing toxic byproducts (if any).It is expected that better understanding of the natural assem-

blages of microbial communities and detailed knowledge ofnaturally occurring symbiosis will represent a lead for biologicallyinspired design of multispecies microbial cell factories to comple-ment current efforts using more conventional genetic engineeringapproaches.

Trends in Biotechnology September 2013, Vol. 31, No. 9heterologous expression and/or hydrogenase engineer-ing for oxygen tolerance or increased activity, inactiva-tion of the uptake [NiFe]-hydrogenase in cyanobacteria,fine-tuning of the oxygen-evolving activity of photosys-tem II (PSII), optimization of the e-flux towards hydrog-enase, and others [17]. However, despite manyencouraging proof-of-principle demonstrations, photo-synthetic hydrogen production remains low for indus-trial purposes.

Host tolerance to high concentration of recombinant-pathway product and/or precursorsIn contrast to ethanologenic microorganisms such as theyeast Saccharomyces cerevisiae or the bacterium Zymo-monas mobilis that tolerate >15% ethanol in the externalmedium, the cyanobacterium Synechocystis sp PCC 6803tolerates only up to 1%. Although such inhibitory concen-trations have not been met from ethanol producing cya-nobacteria [13], it has been shown that imbalancesbetween the recombinant enzymes activities may resultin growth inhibitory concentrations of the toxic interme-diate acetaldehyde [18]. Interestingly, a recent studyshowed improved recombinant-biodiesel yields in E. coliby a dynamic sensorregulator system that adjusts recom-binant enzymes activities according to the levels of thehost key metabolites [19].

mo

into

119f po

68

and

i in Table 1. Current and potential biofuels from cyanobacteria or

Bioproduct Strategy

Cyanobacteria

Ethanol H.E. pdc and adh from Zymomonas mobilisSynechocystis sp. PCC 6803

H.E. pdc from Z. mobilis, overexpressing slrand disruption of the biosynthetic pathway ob-hydroxybutyrate into Synechocystis sp. PCC

Isobutyraldehyde H.E. kivD from Lactobacillus lactis, alsS fromBacillus subtilis, ilvCD from Escherichia coli insertion of extra copies of rbcLS genes inSynechococcus elongatus PCC7942

Isobutanol H.E. kivD from L. lactis and yqhD from E. col

ReviewAssembly of complex enzymesEnzymes such as hydrogenases or nitrogenases contain attheir active sites complex metal centers. Biosynthesis ofthe ironmolybdenum cofactor and assembly of fully func-tional holo-nitrogenase requires at least 15 gene products.Although knowledge on this field has progressively in-creased during recent years, it is still fragmentary[20,21] and limits recombinant pathway design and intro-duction into selected hosts.

General physiological and metabolic adaptationsDuring the course of evolution, some microbes becomeexquisitely specialized to carry out some metabolic path-ways for which they orchestrate sophisticated arrays ofphysiological and metabolic adaptations. Azotobacter

elongatus PCC7942

1-Butanol H.E. hbd, crt, and adhE2 genes from Clostridiumacetobutylicum, ter from Treponema denticola, aatoB from E. coli in S. elongates PCC7942

H.E. bldh from Clostridiumsaccharoperbutylacetonicum, yqhD from E. coli(among others) in S. elongates PCC7942

Isoprene production H.E. ispS from Pueraria montana expressed undregulation of the psbA2 promoter in Synechocyssp. PCC 6803

Fatty acids H.E. a thioesterase, among others modifications,Synechocystis sp. PCC 6803

Fatty alcohols H.E. far from Simmondsia chinensis under thecontrol of Prbc promoter

Hydrogen H.E.[Ni-Fe] hydrogenase hynSL from Alteromonasmacleodii, and Thiocapsa roseopersicina andaccessory genes in S. elongatus PCC7942[FeFe]-hydrogenase hydA1 from Cr inSynechocystis sp. PCC 6803

Insertional disruption of the hupL gene in Nostosp. PCC 7422

Microalgae

Triacylglycerol (TAG) sta6 and sta7-10 starchless mutants of Cr

sta7-10 (isoamylase mutants) complementedstrains

Hydrogen Replacement of wild type promoter of the Nac2gene by a copper repressible promoter in Cr

Mutant strain with a double amino acid substituti(L159I-N230Y) in D1 of Cr

Optimization of the e- flux towards hydrogenasestate transitions mutant6 (stm6) in Cr

aCr, Chlamydomonas reinhardtii; H.E., heterologous expression; u.l.d, unit.litre.day.dified microalgaea

Results/comment Refs

2,ly-03

5.2 mmol ethanol OD730/u.l.d (up to 230 mg/l in 4weeks)212 mg/l.d

[13]

[18]

6230 mg isobutyraldehyde/l.hHigher than cyanobacterial productivities ofhydrogen or ethanol.

[14]

S. 450 mg isobutanol/l in 6 d [14]

Trends in Biotechnology September 2013, Vol. 31, No. 9vinelandii displays a concerted set of mechanisms (in-creased aerobic respiration and exopolysaccharide pro-duction and duplication of housekeeping genes) for itsparticular specialization to run strictly anaerobic path-ways (mainly N2- fixation and H2 metabolism) togetherwith an obligate aerobic life style [22]. Similarly, genetic,biochemical and genomic analyses uncovered unique char-acteristics for the remarkably high ethanol producer Z.mobilis, including a specific metabolic pathway for anaer-obic fermentation of glucose and an array of genetic deter-minants that might account for the exceptionalethanologenic properties [23]. In some other cases, knowl-edge of the genetic basis of some metabolic switches is notavailable to design strategies genetic improvement. Thismight be the case of those targeted bioenergy carriers that

nd14.5 mg 1-butanol /l in 7 d [16]

29.9 mg 1-butanol/l [59]

ertis

50 mg isoprene/g dw.d under high light conditions [60]

in 200 mg/l fatty acids secreted into the culturemedium

[61]

0.2 mg/l fatty alcohols [62]

Accumulation of holo-enzyme that displayedhydrogenase activity in vitro

in vitro-active hydrogenase

[63]

[64]

c 100 mmoles H2/mg chlorophyll a.h (three timeshigher rate than that of the parental strain)

[65]

2- to 10-fold increase in TAG accumulation [6668]

400 mg/l starch exceeding in 4 d (significantlyhigher than those achieved by the parental strains)Higher values in total lipids at 96 h of nitrogendeprivation: 83118 mg lipid/l

[68]

20 mmoles H2/l13.1 mmol/H2 mol. Chl

[69]

on 500 ml H2/l; 5.77 ml/l.d10-fold higher wild type and fivefold CC124 strain,mostly due to a longer production phase

[70]

540 ml H2/l4 ml/h (fivefold higher than the wild type)

[71]

523

constitute the natural carbon reserves of cyanobacteriaand microalgae (carbohydrates or lipids) that normallyaccumulate under unbalanced growth after nutritionalor environmental adverse conditions compromise over-all productivity [24]. The kind of genetic determinantsfor the previous examples are currently difficult toidentify because they used to be encoded by a multiplic-ity of genes that contribute to the trait in an additivefashion; sometimes synergistic or even as emergentproperties of the system. This fact highlights the chal-lenge of optimizing recombinant pathways in hostsselected for other beneficial traits.

Limitation of molecular biology toolkit for cyanobacteriaand microalgaeThis aspect also precludes a faster improvement of strainsby genetic engineering in the short term. Thus, recombi-nant pathwayhost overall incompatibilities may includecombinations of the previously discusses aspects in addi-tion to unforeseen ones.

Natural microalgalbacterial consortia as a source ofbiotechnological insightsIn the wild, microalgae live and have evolved in the contextof multispecies microbial consortia. Beneficial interactionsrange from mutualistic to symbiotic, and their stabilityrelies on two important organizing features: (i) trading ofmetabolites, mainly as crossfeeding but also exchange ofdedicated molecular intra- or interspecific signals; and (ii)specialization and division of labor [25].

In most algaebacteria consortia, microalgae provideoxygen and organic molecules. In natural systems, thealgal release of dissolved organic carbon ranges from 0to 80% of photosynthates and it is 616% in photobioreac-tors [26]. For bioenergy purposes, the latter can be seen asboth a direct bioenergy loss and a potential secondary lossdue to the assembly of spontaneous and unattractive mi-crobial consortia with low or null biotechnological value[27].

By contrast, bacteria could provide a broader array ofsubstances including CO2, other nutrients, vitamins, andgrowth-promoting substances (Figure 2).

Carbon for nitrogen mutualismsAlthough nitrogen is extremely abundant on Earth as N2,the availability of biologically available nitrogen con-strains the productivity of both terrestrial and aquaticecosystems. Nonetheless, N2-fixing symbioses are commonbetween eukaryotes and cyanobacteria or heterotrophicbacteria and represent a remarkable strategy by whichsome organisms can use N2 from the air indirectly inexchange for organic carbon. In contrast to the photosyn-thetic carbon-fixation ability that eukaryotes have ac-quired through endosymbiosis with cyanobacteria duringevolution, no N2-fixing plastids or eukaryotes are known[28]. In the oceans, the best-known N2-fixing symbioses arebetween several species of pennate diatoms and hetero-cyst-forming cyanobacteria. These associations span the

Reviewrange of epibionts (CalothrixChaetoceros) to endosym-bionts (RicheliaRhizosolenia and RicheliaHemiaulus)[29,30]. The cyanobacteria form short chains of vegetative

524cells plus a terminal heterocyst, which is a differentiatedcell for N2 fixation that provides a microaerobic environ-ment to protect nitrogenase from inactivation by the eu-karyotic host oxygenic photosynthesis. Although little isknown about the molecular basis for these interactions, orhow the symbionts are transmitted from generation togeneration [31], there is genome streamlining associatedwith the endosymbiotic species. The closely associatedspecies within the frustule lack ammonium transporters,and one species is the only known cyanobacterium thatlacks glutamate synthase [31]. There appears to be somespecificity between the cyanobacteria and the host genera[32]. A widely distributed marine planktonic unculturedN2-fixing cyanobacterium (UCYN-A) presents a dramaticreduction in genome size and lacks the genes for photosys-tem II (responsible for O2 evolution), RuBisCo (ribulose-1,5-bisphosphate carboxylase-oxygenase for carbon fixa-tion), and the tricarboxylic acid cycle (for aerobic respira-tion and anabolism) [33]. It was further shown that UCYN-A engages in mutualistic relations with a prymnesiophyteexchanging fixed nitrogen for fixed carbon. A similar asso-ciation appears to exist with freshwater diatoms and it isproposed that these rather simple interactions betweensingle-celled organisms might mirror those earlier primaryendosymbiotic events that gave origin to chloroplasts andmitochondria [34].

Carbon for other nutrients or growth-promotingsubstancesIron is an essential element for life and its low bioavail-ability also limits productivity in large areas of the oceans.Alga-associated heterotrophic bacteria belonging to thegenus Marinobacter release the siderophore vibrioferrin(VF) in a carbon for iron mutualistic relationship [35].

A large proportion of microalgae are auxotrophs orfacultative auxotrophs for vitamins, especially B12, andat least some of them can acquire B12 from more or lessspecific interactions with heterotrophic bacteria [27].

In some cases heterotrophic bacteria appear to providephytohormone-like substances (indole-3-acetic acid) to en-hance several microalgal activities. This has been shownfor the effect of coculturing Chlorella spp. with the plantgrowth-promoting bacterium Azospirillum brasilense foran increased pigment and lipid content, lipid variety,carbohydrates, and cell and population size of the micro-algae [3638].

Such associations between unicellular microorganismsmay provide models for means to interact with cyanobac-terial/microalgal metabolism for biotechnological applica-tions.

Biotechnological use of microalgalbacterial consortiaas multispecies microbial cell factoriesThe combined use of algae and bacteria in microbial con-sortia is gaining renewed interest as a biotechnologicalalternative for the enhancement of yields and reducingproduction costs of algal biomass for bioenergy purposes inintegrated bioprocesses together with CO2 and pollutant

Trends in Biotechnology September 2013, Vol. 31, No. 9removal [39,40].The A. brasilense stimulation of Chlorella spp. growth

and lipids and starch accumulation [36,41] represents a

remarkable example of the biotechnological potential ofmicroalgalbacterial consortia pertinent to bioenergy. Al-so, inoculation with the Pseudomonas-related strain GM41increased Synechocystis sp PCC6803 productivity up toeightfold by helping to degrade toxic compounds commonlyfound in polluted water [42].

Another application of microalgalbacterial consortia isthe development of microbial solar cells that comprisephotoautotrophic microorganisms to harvest solar energyand release organic compounds that are used by electro-chemically active microorganisms to generate electricity[43].

Engineering cyanobacterial/algalbacterial multispeciesmicrobial cell factoriesMultispecies microbial consortia can be subjected to tradi-tional genetic engineering or modern synthetic biologyapproaches to improve current applications of microbialconsortia or envision new ones. Synthetic biology relies inanalogies between biological networks and electronic cir-cuits comprising computing reusable parts and connectors(wires). However, both are current major concerns forsynthetic circuits design and intended function in singlecells. As part of a solution, it has been shown how thedistribution of simple computations among the members ofnon-uniform populations would increase exponentially thecomplexity of circuits that could be executed, bypassing theneed for extensive genetic engineering of a single strain,sometimes very difficult to achieve, and the reusability ofthe parts while reducing the wiring requirement [44,45].Thus, application of this principle might be appealing forgenetic engineering of cyanobacteria/algae, for which thegenetic toolkit is limited. This approach may alleviatepressure towards increasing yields of heterologous path-ways in selected expression platforms, for which overallmetabolic coupling (wiring) is weak and/or mostly poorlyunderstood. In turn, the concept of genetic engineering ofmultispecies microbial cell factories might rely on microbeswith outstanding properties as metabolic/production mod-ules, which can be further optimized for the same or othereasier-to-accomplish tasks.

Microbial consortia comprising heterotrophic cells havebeen engineered to disclose some organizational features ofmulticellularity, ecology, and evolution [46], and to pursuea variety of possible applications. Some remarkable exam-ples pertinent to biofuels production from CO2, althoughindirectly, are: (i) the assembly of functional minicellulo-somes by intercellular complementation using a syntheticyeast consortium that divides the metabolic burden ofexpressing high levels of recombinant proteins amongthe members of the consortium; (ii) simultaneous andefficient fermentation of hexoses and pentoses from ligno-cellulose by coculture of engineered strains specialized inthe fermentation of each sugar; and (iii) engineering of twostrains of E. coli that cooperate in the transformation ofxylan into ethanol; while one strain secretes two hemicel-lulases the other uses the released sugars to produceethanol [45,47].

ReviewTwo recent examples of cyanobacteria/algaebacteriaartificial consortia comprising genetically modifiedmicrobes as CO2- or N2-fixing synthetic parts illustratethe potential of this approach for the sustainable produc-tion of biofuels and biomaterials directly from CO2.

Carbon-fixation cell factoriesThe cost of the carbon source in commercial fermentationscan be as much as 3050% of the overall operating cost, andhence the interest in developing photosynthetic microbial-cell factories primarily based on cyanobacteria or micro-algae. Recent progress in the heterologous expression ofthe E. coli sucrose permease cscB in the cyanobacteriumSynechococcus elongatus has allowed the irreversible ex-port of sucrose into the medium at concentrations >10 mMwithout culture toxicity. Remarkably, the sucrose-export-ing cyanobacterium exhibits increased biomass productionrates relative to the wild type strain, and enhances photo-system II activity, carbon fixation, and chlorophyll content.Additional mutations to minimize carbon flux towardscompeting glucose- or sucrose-consuming reactions furtherimproves sucrose production up to 80% of total biomass.Such a strain/strategy may be a viable alternative to sugarsynthesis by terrestrial plants, including sugar cane [48].Similarly, S. elongatus transformed with Z. mobilis invAand glf genes, encoding invertase and a glucose/fructosefacilitator, respectively, excreted sugars into the mediumin such a way that it supported E. coli growth in theabsence of supplementation with a carbon source [49].Metabolic coupling of photosynthetic modules to E. coli,for which advances in genetic engineering and industrialapplications have few (if any) rivals, is of remarkableimportance. Coculture of sugar-excreting cyanobacteriawith any other second engineered microbe could yield adesired product without a reduced-carbon feedstock insituations where synthesis of the product is incompatiblewith cyanobacterial metabolism (Figure 1A). Moreover,attempts to introduce wild type cyanobacteria as syntheticchloroplasts into animal cells were successful as a proof-of-principle. However, calculations indicated that even if thesugar excreting strains were introduced, a significantlyhigher ratio of bacterial-to-animal cells would be requiredto provide an adequate supply of sugar to the heterotrophichost [50].

Nitrogen-fixing cell factoriesLarge-scale culture of microalgae, which have an averagecomposition of CH1.7O0.4N0.15P0.0094, might represent ahigh-nitrogen-intensive bioprocess, if wastewater and/orother alternatives are not used as partial or completesubstitutes for nitrogen fertilizers [5153]. Although pro-motion of symbiotic N2 fixation represents a remarkablestrategy in agriculture, the sophisticated interplay of rec-ognition signals that underlay most known N2-fixing sym-biosis [28] has complicated the development ofexchangeable N2-fixing parts for synthetic biologyapproaches.

Most free-living diazotrophs normally fix enough N2 fortheir needs and excrete low to undetectable amounts of N2-fixation products into the medium. Recently, it was shownthat disruption of the genetic system signaling the nitrogen

Trends in Biotechnology September 2013, Vol. 31, No. 9status in an Az. vinelandii mutant strain by inactivation ofthe nifL gene, expresses nitrogenase constitutively andexcretes ammonium into the surrounding medium. Con-

525

C-intput(A) C-output

Reviewversely to wild type Az. vinelandii, the ammonium-excret-ing strain engaged in carbonnitrogen mutualistic rela-tions with the oleaginous microalgae Chlorellasorokiniana, Pseudokirchneriella sp., and Scenedesmus

(CO2)

(B)

(C)

Photosynthecmodule

Produconmodule

CO2

CO2 N2 H2

H2

H2-producingmodule

Photosynthecmodule

Hydrogen

Air

H2O O2

CaHbOc

CO2

NH4+

H2OO2

CaHbOc

CO2H2O

O2

CaHbOc

N-xingmodule

C-xingmodule

(C-biofuels)

TRENDS in Biotechnology

Figure 1. Conceptual diagrams of multispecies microbial cell factories. (A) General

overview of the principle consisting in the metabolic compartmentalization

between a specialized photosynthetic module cross-feeding organic carbon,

likely simple sugars (CaHbOc), and O2 to a production module for carbon-

containing biofuels with some CO2 recycling. Although this is a widespread

metabolic loop in natural communities, recent improvement by genetic

engineering has represented valuable proof-of-concept suggesting that it is

worth further development of this principle for biotechnological purposes. (B)

Model of carbonnitrogen exchanging synthetic multispecies microbial cell factory

that would allow the production of biomass from carbon and nitrogen from the air

as an alternative platform to diazotrophic cyanobacteria. A recent demonstration

showed how rather simple genetic engineering manipulations can make microbes

more prone to engage in mutualistic relations that would reduce the cost of

biomass production and provide a platform with metabolic spatial

compartmentalization to facilitate further optimization of specific pathways. (C)

Hypothetical model derived from (B) for the nitrogenase-dependent production of

H2. A few microbes such as Azotobacter vinelandii produce H2 in the air by means

of nitrogenases. In the absence of N2, more electrons are diverted to the reduction

of H+ by nitrogenases. Although we are not aware of such a demonstration, we

anticipate that both the available knowledge on N2 and H2 metabolisms and

molecular tools for genetic manipulation of Az. vinelandii would make it possible

to challenge this hypothesis in the near future.

526(A)

(B) (C)

(D) (E)

O2

O2O2

O2

O2N2

N2

N2

CO2

CO2

Vitamin B12Algae

DOM

DOM

Siderophore

CO2CO2

CO2Diatom frustule

Metabolites

Heterocyst

Chloroplast

Trends in Biotechnology September 2013, Vol. 31, No. 9obliquus [51], when no sources of carbon or nitrogen otherthan air were supplemented into the medium. Inoculationwith the ammonium-excreting strain mimicked microalgalgrowth equivalent to an ammonium amendment of up to0.5 mM. In these artificial symbioses, although the numberof viable bacterial cells decreased dramatically for the wildtype strain, it remained more stable for the ammonium-excreting cells, suggesting that it was engaged in a morerobust symbiotic relation with microalgae. Mostly micro-algal cells proliferated during coculture, therefore, theresulting biomass mirrored the composition of that ofthe oleaginous microalgae, which attained lipid contentsof up to 30% on a dry biomass basis [51]. Both the metabolicpathways for biological N2 fixation and triacylglycerolaccumulation are rarely found in any single native micro-bial strain [54], and would likely be difficult to attain bygenetic engineering of a single strain [55]. In addition, thisN2-fixing module showed an apparently low selection ofpartner because it could be metabolically coupled withdiverse wild type microalgae or cyanobacteria. This casestudy provides proof-of-concept for the development of N2-fixing modules that might be coupled to photosynthetic andsugar-producing synthetic cellular parts for enhanced bio-mass production and a metabolically compartmentalized

DinoagellateFe

FeFe Fe

TRENDS in Biotechnology

Figure 2. Natural oceanic N2-fixing algalbacterial interactions. (AC) Carbon

nitrogen mutualistic interactions. (A) Interaction between a diatom and

endosymbiotic filamentous heterocyst-forming (specialized cell for N2 fixation).

(B) Atelocyanobacterium thalassa (UCYN-A) and as yet unidentified

prymnesiophyte similar to Braarudosphaera bigelowii. (C) Endosymbiotic

filamentous cyanobacteria and chain forming diatoms (adapted from [29]). (D)

Carboniron mutualistic interaction between the dinoflagellate Scripsiella

trochoidea and the bacterium Marinobacter sp. strain DG879 [35]. (E) CarbonB12vitamin mutualistic relation between the green microalgae and the bacteria of the

order Rhizobiales [27]. Abbreviation: DOM, dissolved organic matter.

platform (Figure 1B). Az. vinelandii, already proposed asan alternative host for the expression of anaerobic path-ways [22], might represent an alternative host underconsideration for hydrogen production and possibly otherO2-sensitive pathways (Figure 1C).

Living Az. vinelandii cells have been successfully intro-duced into either plant or microalgal cells to constructsynthetic N2-fixing endosymbiotic systems [56], makingfuture steps for introducing optimized N2-fixing modulesinto eukaryotic cells feasible.

Several filamentous heterocyst-forming cyanobacteriamay also serve as carbon- and N2-fixing metabolicallycompartmentalized platforms. In this case the carbon-and N2-fixing modules would be inseparable, which wouldbe only useful for some potential applications. Neverthe-less, it would be desirable to encourage the development ofthe required genetic tools to bring into play these naturallycompartmentalized metabolic platforms [57].

Additional possibilitiesIndustrial production of biofuels and raw materials wouldrequire large scale nonaxenic cultivation of microorgan-isms, probably in open ponds. This fact would open addi-tional possibilities for synthetic consortia, for example, tocontrol microbial competitors or grazers for crop protection[6,10] by engineered microbes producing specific antimi-

Reviewcrobials similarly as has been shown for biomedical appli-cations [47]. Engineered bacteria can also be introduced toprovide or amplify signals triggered by expensive chemicaleffectors, to aid in the utilization of recalcitrant nonexpen-sive nutrients or stress tolerance, to facilitate harvesting ordownstream processing.

Box 4. Outstanding questions

The most significant aspects that need to be addressed to advancethe field should be approached multidisciplinary: Synthetic biology aspects

The genetic toolkit for nonconventional microbes (other than E.coli or yeast) naturally efficient to perform specific tasks needs tobe improved and shared among public and private sectors. Morespecific cases that would benefit from the used of syntheticmicrobial consortia should be identified to stimulate creativethinking on synthetic circuits design. The real challenge in designwould be to account for all the aspects below.

Cultivation strategiesPhysical separation of the consortium partners might not be

practical for large-scale cultivation. Thus, detailed characterizationof prototype synthetic consortia including nutritional and cultiva-tion requirements is needed. This aspect should be developedback-to-back together with pilot-scale ponds/photobiorreactorsdesign. When physical insulation cannot be bypassed, syntheticbiology designs for self structured microbial communities andengineering alternatives for inexpensive reactors constructionmight be considered.

Life cycle analysisThe previous activities should complete the set of data to assess

the economic and environmental sustainability and benefits of thealternative use of different synthetic consortia for biofuels or bulkchemicals production for a variety of hypothetical scenarios.

Ecological riskAs for any other genetically modified organism, the ecological

risk associated to its liberation to the environment, even in a

semicontained way as algal culture would be, should be modeledin advance so as to be taken into account while consideringalternative designs [11,72].Beyond the biotechnological potential of artificial micro-bial consortia, much work is left to optimize custom-madeparts to fit a variety of purposes. Detailed knowledge ofnaturally occurring symbiosis might represent a lead forbiologically inspired design without resigning the innova-tion capacity [58].

Concluding remarksProof-of-concept demonstration of carbon- or nitrogen-re-leasing modules may catch the attention of more research-ers to develop and apply the concept of multispeciesmicrobial cell factories. Much work is ahead to continueexploring its potential as well as to identify specific draw-backs (Box 4). Consideration of this alternative might alsohave some influence on strain selection and bioprospectingbecause bioresources that are not attractive as single straincell factories might be valuable as metabolic modules. Thus,the multispecies microbial cell factories concept is a prom-ising alternative to long-standing problems in genetic engi-neering of complex metabolic pathways to improvesustainable production of bioenergy and other commodities.

AcknowledgementsMany pioneering and/or relevant articles could not be cited because ofspace limitations. We are very grateful to the editor and reviewers forconstructive and insightful comments. This work will be included in thePh.D. thesis of J.C.F.O-M (UNMdP). J.C.F.O-M and M.D.N. are fellowsand L.C. is a career researcher at the CONICET, Argentina. Supported byANPCyT Grants PICT-2007 01717 and PICT-2011 2705, CONICETGrant PIP 2012-2014 N8 1032 and FIBA. This work is dedicated to thememory of Julio Curatti, who passed away on February, 27th, 2013.

Disclaimer statementThe authors declare no conflict of interest.

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Genetic engineering of multispecies microbial cell factories as an alternative for bioenergy productionPhotosynthetic microbes for the production of bioenergy and biomaterialsProgress and constraints of genetic engineering of photosynthetic microbial cell factoriesIncompatibility of oxygenic photosynthesis with anaerobic metabolismHost tolerance to high concentration of recombinant-pathway product and/or precursorsAssembly of complex enzymesGeneral physiological and metabolic adaptationsLimitation of molecular biology toolkit for cyanobacteria and microalgae

Natural microalgal-bacterial consortia as a source of biotechnological insightsCarbon for nitrogen mutualismsCarbon for other nutrients or growth-promoting substances

Biotechnological use of microalgal-bacterial consortia as multispecies microbial cell factoriesEngineering cyanobacterial/algal-bacterial multispecies microbial cell factoriesCarbon-fixation cell factoriesNitrogen-fixing cell factoriesAdditional possibilities

Concluding remarksDisclaimer statementReferences