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New challenges in mic oalgae bio echnology
Fede ico Val e dea,*, F ancisco J. Rome o-Campe oa,b, Rosa Leónc, Miguel G. Gue e oa
and Au elio Se anoa,*
aIns i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa y CSIC, A . Amé ico Vespucio 49,
41092 Se illa, Spain; bDepa men o Compu e Science and A i icial In elligence, ETSII, Uni e sidad de
Se illa, A . Reina Me cedes s/n, 41012 Se illa, Spain; c
Á ea de Bioquímica - Depa amen o de Química y
Ciencia de los Ma e iales, Facul ad de Ciencias Expe imen ales, Uni e sidad de Huel a, A . de las Fue zas
A madas, 21071 Huel a, Spain.
____________
*Co esponding au ho s:
E-mail add esses: ede ico. al e de@ib .csic.es (F. Val e de) and au elio@ib .csic.es (A. Se ano)
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Abs ac
Pho osyn he ic p o is s, also called mic oalgae, ha e been sys ema ically s udied o mo e han a
cen u y. Howe e , only ecen ly b oad bio echnological applica ions ha e os e ed a no el wa e o esea ch
on hei po en iali ies as sus ainable esou ces o enewable ene gy as well as aluable indus ial and ag o-
ood p oduc s. A he ecen VII Eu opean Cong ess o P o is ology held in Se ille, h ee ou s anding
examples o di e en esea ch s a egies on mic oalgae wi h bio echnological implica ions we e p esen ed,
which sugges ed ha in eg a i e app oaches will p oduce e y signi ican ad ances in his ield in he nex
u u e. In any case, in ense esea ch and he applica ion o sys ems biology and gene ic enginee ing
echniques a e absolu ely essen ial o each he ull po en ial o mic oalgae as cell- ac o ies o bio-based
p oduc s and, he e o e, could con ibu e signi ican ly o sol e he p oblems o biosus ainabili y and ene gy
sho age.
Keywo ds: Algae bio echnology; Bio uels; Gene ic enginee ing; Mic oalgae; Pho osyn he ic p o is s;
Sys ems biology
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In oduc ion
Ene gy and i s sus ainable p oduc ion is one o he mos impo an esou ces o mankind. Sunligh is
by a he mos impo an inpu o ene gy o Ea h, and pho osyn hesis is he main biological p ocess
channeling sola ene gy in o he biosphe e. Euka yo ic mic oalgae a e a axonomically b oad and
he e ogeneous g oup o pho o ophic p o is s o inc easing bio echnological in e es due o hei highe
pho osyn he ic e iciencies ela i e o land plan s (mic oalgae con ibu e up o 25% o global pho osyn he ic
p oduc i i y), ele a ed g ow h a es and as me abolic capabili ies (Ra en and Falkowski 1999). In
pa icula , he g een mic oalgae (Chlo ophy a) sha e he same pho osyn he ic machine y as he highe plan s,
acco ding o hei close phylogene ic ela ionships.
Mic oalgae such as Chlo ophyceae and Bacilla iophyceae use sunligh ene gy and a simple se o
abundan , cheap esou ces (ca bon dioxide, wa e and mine als) o gene a e a po en ial la ge numbe o
aluable p oduc s o echnological in e es . These p oduc s can be applied, ei he di ec ly o a e
ans o ma ion, in indus ial, pha maceu ical and ag o- ood p ocesses; examples a e ca o enoids, oils,
polysaccha ides, pigmen s, bioe hanol, hyd ogen, mic oalgal biomass (León e al. 2008; Finazzi e al. 2010;
Cado e e al. 2012). Indeed, some mic oalgal species accumula e impo an amoun s o hese compounds
unde speci ic en i onmen al condi ions, so his bio echnologically ele an pheno ypic ea u e is amenable o
op imiza ion by gene ic enginee ing app oaches (León e al. 2008, Cado e e al. 2012).
This e iew summa izes he con ibu ions p esen ed by h ee mic oalgal bio echnologis s a he
symposium New Challenges in Mic oalgae Bio echnology held du ing he VII Eu opean Cong ess o
P o is ology, which was o ganized o he i s ime as a join mee ing in pa ne ship wi h he In e na ional
Socie y o P o is ologis s (VII ECOP – ISOP Join Mee ing) in Se ille, Spain, 5-10 Sep embe 2015. The use
o mic oalgae as sus ainable oil sou ces o bio uels will be e alua ed and discussed in he i s sec ion, gi en
ha some o hese pho o ophic p o is s in insically accumula e high oil le els (up o mo e han 80% o he
d y weigh ). Signi ican ecen ad ances in he de elopmen o gene ic manipula ion ools, aimed o imp o e
bio echnological ea u es o mic oalgae as sou ces o enewable esou ces, a e p esen ed in he second
con ibu ion. Finally, he applica ion o compu a ional modeling as a sys ems biology s a egy o be e
unde s and mic oalgal me abolic and cell signaling ne wo ks will doub less con ibu e o disco e no el
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p ope ies wi h ele an bio echnological implica ions, as is p esen ed in he hi d con ibu ion. In any case, i
is clea ha in ense esea ch and he applica ion o gene ic enginee ing a e absolu ely essen ial o each he
ull po en ial o mic oalgae as cell ac o ies and, he e o e, will signi ican ly con ibu e o sol e he p oblems
o biosus ainabili y and ene gy sho age.
Bio uel om mic oalgae?
Mic oalgae a e a polyphyle ic g oup and a huge pool o biological di e si y. P ope ies ypical o
highe plan s a e combined in mic oalgae wi h bio echnologically amenable a ibu es o mic obial cells.
These and o he p ope ies o mic oalgae (such as hei me abolic plas ici y, ole ance o ex eme
en i onmen al condi ions and amenabili y o gene ic enginee ing), a e aluable o bioindus y. These
pho osyn he ic mic oo ganisms a e a sou ce o compounds wi h comme cial alue, such as ca o enoids,
phycobilip o eins, polyunsa u a ed a y acids, polysaccha ides and an a ay o bioac i e compounds o
ag icul u e and ood, eed, pha maceu ical, cosme ic, and chemical indus ies. Mic oalgae can also be o use
in he eco e y o was ewa e and in aba emen o ca bon dioxide.
Mic oalgae ha e been p oposed as an al e na i e sou ce o enewable bio uel, capable o mee ing
he global demand o anspo uels. Al hough he “mic oalgae o bio uel” concep was i s sugges ed in he
1940s, i has ecen ly ecei ed new a ac ion and suppo . A seminal a icle by Yusu Chis i published in
2007 (Chis i 2007) has been pa icula ly e ec i e in d awing he a en ion o esea che s and in es o s. In his
way, many esea ch g oups we e a ac ed o he ield, oge he wi h comme cial en u es es ablished
he ea e . Acco ding o da a a ailable in Thomson Reu e s’ Web o Science™, he numbe o published
i ems pe yea on “bio uel om mic oalgae” has g own exponen ially, om less han 5 be o e 2007 o o e
390 in 2014.
A en ion on mic oalgae o bio echnological easons has also bene i ed om he ac ha mass
p oduc ion o liquid bio uels om plan biomass is being inc easingly ques ioned. The “ ood e sus uel”
dilemma and he limi a ions in a ailable e ile land o a wo ld’s g owing popula ion a e easons o
econside he bio uel p oduc ion om c op plan s (Sea chinge e al. 2015). Mic oalgae ep esen an
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al e na i e o land plan s, since cul u es could be de eloped in non-a able land, employing b ackish, saline o
e en was e wa e , as well as ca bon dioxide om lue gases as ca bon sou ce. Values o expec ed uel
p oduc i i y a ound 20,000 L pe hec a e and yea seem easonable o ou doo cul u e o mic oalgae (Moody
e al. 2014), al hough some subs an ially highe p ojec ions a e equen ly a gued in he li e a u e. Howe e ,
mos o he p ojec ed alues o igina e om g oss ex apola ions, bo h in a ea and ime, om sho - e m ials
in small size acili ies, i no di ec ly om labo a o y expe imen s. Analogous conside a ions apply o
published li e cycle assessmen s and o p oduc ion p ices app aisals o ei he biomass o bio uel om
mic oalgae. The escala ion o hese p ocesses o e s a e y challenging subjec o applied esea ch.
Up o now, sca ci y o scien i ic and echnical knowledge, as well as limi ed p ac ical expe ience,
de e mines a high p ice o mic oalgal biomass and he bio uel he eo . The lowes p oduc ion cos in
comme cial algae p oduc ion seems o be abou US$ 4–5 pe kg algal biomass. Signi ican R&D e o s a e
cu en ly being add essed o he de elopmen o iable p ocesses able o massi ely gene a e mic oalgal
bio uels a p ices ha can compe e wi h hose o es ablished uels (Sing e al. 2013). The p oduc ion s ep has
o be conside ably imp o ed, bu also ha es ing, biomass d ying and ex ac ion o bio uel p ecu so and i s
con e sion in o he inal p oduc s ill need subs an ial op imiza ion.
Selec ion o he mos app op ia e mic oalgal s ains is a key issue (Figu e 1). No jus he con en o
he bio uel p ecu so (ei he e men able suga s o a y acids) should be conside ed, bu a he he p oduc ion
capaci y, looking o he op imal combina ion o p oduc le el and biomass p oduc i i y. The con inuous
cul u e app oach is he mos app op ia e me hodology o he sc eening o mic oalgae o he pu pose o
bio uel p oduc ion, as i allows he de e mina ion o eal p oduc i i y o a pa icula bio uel p ecu so (Del
Río e al. 2015). Also c ucial in he selec ion o he s ain is he abili y o de elop ou doo s as a monoalgal
cul u e h oughou he yea . Many expec a ions a e placed on he po en ial o gene ic enginee ing o he
gene a ion o s ains wi h supe io p oduc i i y o ei he a y acids o e men able ca bohyd a es, bu u he
de elopmen o no el echniques o e icien manipula ion o mic oalgae is s ill needed.
P oduc ion o bio uels is la gely policy-d i en and i s p o i abili y has been ques ioned, e en a oil
p ices abo e US$ 100/ba el (bbl). Cu en a e age p ice o c ude oil is a ound US$ 50/bbl, and i is
expec ed o inc ease up o abou US$ 90/bbl by 2025 (Wo ld Bank G oup 2015). Wi hin his amewo k i

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does no seem ha con en ional bio uels ha e an easy way o de elop in he nea u u e, wo s s ill when
conside ing hose om mic oalgae. In such a scena io and in o de o compe e wi h oil a cu en p ices, he
p oduc ion p ice o mic oalgal biomass con aining 25% oil should be a ound US$ 0.1/kg. A such p ices,
p o ein- ich mic oalgal biomass would compe e a o ably wi h o he p o ein sou ces, such as soybean
(cu en p ice, US$ 0.4/kg).
The ques ion he e o e is, does i make sense o use mic oalgal biomass o uel o a he as
ood/ eed?
De elopmen o new molecula ools o gene ic enginee ing o euka yo ic mic oalgae
In he las yea s, he e has been an inc easing in e es on he gene ic enginee ing o mic oalgae, as a
po en ial ool o economically easible p oduc ion o bulk ma e ials and o enhance p oduc i i y o high-
added compounds (Wij els e al. 2010). Howe e , ou ine gene ic manipula ion has been limi ed o a ew
species un il ecen ly. The lack o sui able p omo e s and o he egula o y sequences a e, besides low
e iciency and ins abili y o ansgenes exp ession, he main di icul ies p e en ing nuclea ans o ma ion o
new mic oalgal s ains (León e al. 2007). Since low exp ession o exogenous genes is hampe ing he e icien
enginee ing o me abolic pa hways and he use o mic oalgae as pla o ms o he p oduc ion o ecombinan
p o eins, i is necessa y o de elop new ools, which ensu e s abili y and high exp ession le els o he
ansgenes.
He e, we p opose a new me hod o exp ess ansgenes in mic oalgae: co- ans o ma ion wi h wo
naked p omo e -less genes, a selec able an ibio ic- esis an gene and he gene o ou in e es (Figu e 2). These
genes a e andomly inse ed in o he nuclea genome so ha hei ansc ip ion elies on hei adequa e
inse ion in a egion adjacen o an endogenous genomic p omo e o in ame wi h a na i e gene. This
app oach is especially app op ia e o ans o m mic oalgal species o which no endogenous p omo e s o
speci ic exp ession plasmids ha e been designed. The ac ha he ansgenes a e exp essed unde he con ol
o endogenous p omo e s educes he isk o silencing e en s and hei in eg a ion in o he genomic
en i onmen o he p omo e gua an ees he p esence o enhance s, ansc ip ion ac o s o o he egula o y
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egions essen ial o he adequa e exp ession o he ansgene. A p omo e -less co- ans o ma ion app oach
has been success ully used o exp ess yeas locculins in Chlamydomonas einha d ii in o de o ob ain
ansgenic mic oalgal s ains wi h highe sel - loccula ion abili y han he con ol o un ans o med ones
(Díaz-San os e al., 2015). Floccula ion is e y impo an om he enginee ing poin o iew i we conside
ha mic oalgal ha es ing can make up 30% o he o al cos o algal biomass p oduc ion (Salim e al. 2012).
Howe e , cloning o locculins has been limi ed by hei oxici y o bac e ia caused by hei excessi e leng h
and la ge numbe o andem epea s in hei cen al domain. P omo e -less co- ans o ma ion a oids he need
o cloning because la ge amoun s o DNA om he desi ed gene can be di ec ly ob ained by ampli ica ion o
by a i icial syn hesis and inse ed in he genome o he hos s ain.
We ha e designed also a plasmid o he ansla ional usion o he gene o in e es wi h a selec able
an ibio ic- esis an gene, whe e he p o ein o in e es and he p o ein con e ing esis ance o he an ibio ic,
used by a sel -clea ing pep ide (De Felipe e al. 2006), a e p ocessed om he same polyp o ein. Sc eening
ans o man s wi h inc easing amoun s o he selec i e an ibio ic p o ides a simple me hod o selec ing
clones wi h he highes exp ession le el o he selec able ma ke gene and, consequen ly, o he gene o
in e es ; u he mo e, main aining he ans o man s unde selec i e condi ions imp o es he s abili y o he
ansgenes.
Al hough much wo k is s ill necessa y, hese new molecula ools will allow he imp o emen o
ansgene exp ession in mic oalgal nuclei and he gene ic modi ica ion o new species o indus ial in e es .
A s udy on he basic helix-loop-helix ansc ip ion ac o amily in Chlamydomonas
einha d ii using he gene co-exp ession ne wo k ChlamyNET
The pho osyn he ic p o is Chlamydomonas einha d ii is he mos impo an model o ganism o
unicellula g een algae (Ha is, 2001; Hanikenne, 2003; Ma suo and Ishiu a, 2011; Sla eyko a e al., 2016).
Recen ly, Chlamydomonas has a ac ed a en ion due o i s po en ial bio echnological applica ions (K use and
Hankame 2010; Si akuma e al. 2010). In o de o cha ac e ize di e en Chlamydomonas s ains and hei
esponse o di e en condi ions, a massi e amoun o 'omics' da a has been p oduced (Cas ui a e al. 2011;
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Gonzalez-Balles e e al. 2010; Mille e al. 2010; U zica e al. 2012). In o de o in eg a e hese da a and
gene a e sys emic and global cha ac e iza ions, a i s app oach based on molecula sys ems biology has been
aken (Dal'Molin e al. 2011; Lopez e al. 2011; Rome o-Campe o e al. 2013; Zheng e al. 2014). In his
s udy, we used ChlamyNET (Rome o-Campe o e al. 2015), a gene co-exp ession ne wo k ha in eg a es
RNA-seq da a, o analyze he basic helix-loop-helix (bHLH) ansc ip ion ac o (TF) amily in
Chlamydomonas. Speci ically, using ChlamyNET, we ha e de e mined he biological p ocesses po en ially
egula ed by bHLH TFs and he DNA sequences ecognized by hem.
The bHLH ansc ip ion ac o amily o plan s is cha ac e ized by he p esence o a conse ed
p o ein domain consis ing o wo α helices connec ed by a loop and iden i ied in he P o ein amily (P am)
da abase wi h he id PF00010. Recen genomic analyses ha e iden i ied eigh bHLH TFs in he
Chlamydomonas genome (Pé ez-Rod íguez e al. 2010). Se en o hese ansc ip ions ac o s exhibi
signi ican gene co-exp ession pa e ns and can be iden i ied in ChlamyNET using i s sea ch u ili y and he
bHLH P am id PF00010, as shown in Figu e 3A. These TFs cons i u e h ee di e en clus e s (Figu e 3B).
The bigges clus e includes he bHLH genes C e14.g620850, C e01.g011150, g4643 and g4645 and i is
loca ed a he co e o ChlamyNET. bHLHs a e co-exp essed wi h a signi ican ly high numbe o genes, which
makes hem hub genes in he ne wo k playing key oles in he ansc ip ome obus ness and in o ma ion
p ocessing.
In o de o de e mine he po en ial biological p ocesses egula ed by bHLH TFs in Chlamydomonas,
we pe o med a Gene On ology (GO) e m en ichmen analysis o e he genes co-exp essed wi h hem using
ChlamyNET. We iden i ied ansmemb ane anspo and ca bohyd a e me abolism as he wo mos
signi ican biological p ocesses po en ially egula ed by he bHLH TFs in Chlamydomonas (Figu e 3C). Fo
ins ance, he genes C e02.g110800 and C e13.g569850 ha codi y o ni a e and ammonium anspo e s
espec i ely a e highly co-exp essed wi h he bHLH TF C e01.g011150. This bHLH TF is also highly co-
exp essed wi h genes codi ying o p o eins in ol ed in ni ogen me abolism such as he ni i e and ni a e
educ ases, C e09.g410750 and C e09.g410950, espec i ely. A he same ime, he genes C e08.g384750 and
g3160 ha codi y o an alpha-amylase and an isoamylase espec i ely a e highly co-exp essed wi h he
bHLH TF C e14.g620850.
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In o de o de e mine DNA sequences po en ially ecognized by he bHLH TFs in Chlamydomonas
we analyzed, using ChlamyNET, he p omo e sequences o he genes co-exp essed wi h hem. This analysis
e ealed ha he E-box sequence and one o i s a ian ecognized by he PIF5 TF in A abidopsis haliana
ac ually appea ed in he p omo e s o many genes highly co-exp essed wi h bHLH TFs (Figu e 3D). These
DNA sequences a e p esen , o ins ance, in he p omo e s o he genes C e02.g110800 and C e13.g569850
ha codi y o ni a e and ammonium anspo e s espec i ely and in he p omo e o he gene
C e08.g384750 ha codi y o an alpha-amylase.
Summa izing, he analysis using ChlamyNET sugges s ha he amily o bHLH TFs plays a key ole
in he egula ion o ele an biological p ocesses in Chlamydomonas physiology such as ansmemb ane
anspo and ni ogen/ca bon me abolism. This egula ion seems o be exe ed h ough ecogni ion o E-
boxes and simila DNA sequences loca ed in he p omo e o po en ial a ge genes. These in silico
p edic ions should be aken as a hypo hesis ha needs in i o and in i o alida ion.
P ospec s
In o de o cope wi h he high expec a ions c ea ed in he mic oalgal bio echnological ield, a numbe
o challenges will need o be add essed in he nea u u e. In his e iew we ha e shown a small sample o
hese solu ions; o he impo an aspec s ela ed o he gene ic manipula ion o algae and upscaling o he
labo a o y expe imen al biomass p oduc ion ials o la ge, indus ial ins alla ions, will be needed.
As new ecological niches a e p ospec ed and new massi e big-da a echniques a e employed, he
numbe o accoun ed pho osyn he ic p o is s is ising. Today we know some 50,000 mic oalgal species, bu
es ima es sugges mo e han 500,000 species a e sp ead o e he sui able habi a s on Ea h (Cado e e al.
2012). In o de o aise he numbe o isola es and inc ease he chance o ge ing in e es ing new mic oalgal
bio ypes wi h no el enhanced cha ac e is ics o bio uel p oduc ion o wi h a e me abolic ades, he esea ch
in o phylogenomics will ce ainly g ow in he ollowing yea s. This a ea o s udy is bound o b ing su p ises
and open up new ields o applied esea ch, as he mis s ha co e he huge ecological impo ance o his
he e ogeneous g oup o p o is s will be un eiled.
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Figu e 2
Selec ablegene
Geneo in e es
BLE APHVIII
APHVIII
BLE bleomycinbindingp o ein om
S ep oallo eichushindus anus
=
==
=
aminoglycoside3´phospho ans e ase om
S ep omyces imosus
Selec ablegeneSelec ablegene
Geneo in e es Geneo in e es
BLE APHVIIIBLE APHVIIIAPHVIII
APHVIIIAPHVIII
BLEBLE bleomycinbindingp o ein om
S ep oallo eichushindus anus
=
==
=
aminoglycoside3´phospho ans e ase om
S ep omyces imosus
aminoglycoside3´phospho ans e ase om
S ep omyces imosus

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Figu e 3