This article isThis article by protected copyright. All rights reserved. 10.111 doi: differences to lead between the of this version cite thisarticle and asVersion Record.Please copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been microalgae, Keywords: Kingdom *Corresponding author: 4QD, UK 4 3 Kingdom 2 1 Beacham D’AdamoSarah Triterpenoid Engineering The Unicellular Alga Article :Research Article type : (Orcid ID 0000 MATTHIJS MICHIEL DR. AcceptedBiosciences, CollegeofLifeandEnvironmental UniversityExeterSciences, ofExeter, EX4 Rothamsted Research, Harpenden, AL52JQ, United Kingdom PML: Laboratory, Eden Algenuity, MK43United9ND Kingdom Stewartby, Article Plymouth MarineLaboratory
2 +44 1234765773, , Andrew Landels, Andrew
1/pbi.12948 t riterpenoid biology biosynthesis, natural product,algalsynthetic lupeol, betulin,bluebiotechnology
Production 1 , Gino Schiano di Visconte Schiano , Gino
Eden Laboratory/Broadmead Rd,Bedford Laboratory/Broadmead Eden MK43 9ND,United
2 [email protected] , 3 ,
for publication andundergone fullpeer review buthasnot Michael J Michael
Phaeodactylum , Prospect - 0002 .
Allen - 1 6051 , Gavin Lowe
Place, TheHoe,Place, Plymouth,PL1 3DH 2 ,4 -
3671) , Andrew Spicer , Andrew
t ricornutum
1
, Joanna Szaub , Joanna
For High For 1 , Michiel Matthijs
- - Newton Value , diatoms Plant 1 , , United United , Tracey Tracey 1 *
, This article isThis article by protected copyright. All rights reserved. in undertaken engineering pathway extensive in grown pathway. biosynthesis sterol and mevalonate the Medicago the in enzymes plant three tricornutum Phaeodactylum t the yeasts and bacteria as such T cost their and levels production low their include commercialization triterpenoid for challenges promise as potential demonstrated defense plant development, compounds organic of class diverse a constitute triterpenoids Plant ABSTRACT acids CPR, monooxygenase; OSC, Synthase; LjLUS, pathway; Abbreviations Acceptedth produce to attempts herefore, Article
L. japonicus L. - fetv pu effective
riterpenes as a pharmaceutical precursor pharmaceutical a as
a
truncatula 550 B, Betu BA, : oxidosqualene
L oxidosqualene cyclase oxidosqualene
iiain rm h cmlx itrs rsn i ter aua hosts natural their in present mixtures complex the from rification pilot scale scale pilot ou japonicus Lotus betulin
cytochrome P450 along with its native re native its with along P450 cytochrome cytochrome b5 reductase b5 cytochrome ii acid; linic
n is rcro lupeol precursor its and , a , have attracted great interest. great attracted have hraetcl. n eape s b is example One pharmaceuticals. photobioreactor micro cyclase; IPP, Isopentyl Pyrophosphate; CYP, Pyrophosphate; Isopentyl IPP, cyclase; unicellular eukaryotic unicellular and
ese uel ytae ALS Aaioss hlaa Lupeol thaliana Arabidopsis AtLUS, synthase; Lupeol
alga: alga: niomna interaction environmental MVA,
compounds in industrially relevant microbial systems microbial relevant industrially in compounds
perturbed for the treatment of certain cancers and HIV. Major HIV. and cancers certain of treatment the for a
ou japonicus Lotus M T evalonate he best he a diatom a facility. To our knowledge, this is the most most the is this knowledge, our To facility. ; lcPUFA, lo lcPUFA, ;
the alga. mRNA mRNA performing strains were selected were strains performing
and the first the and
P Here we report the production of of production the report we Here in This was achieved by introducing introducing by achieved was This twy MP non MEP, athway;
ng chain polyunsaturated fatty polyunsaturated chain ng expression levels of the native the of levels expression oxidosqualene cyclase and a a and cyclase oxidosqualene the ductase. ductase. . Several triterpenes have have triterpenes Several etulin photosynthetic that play a play that time that a that time , The introduction of introduction The
which has shown shown has which cytochrome P450 cytochrome
major role in role major - mevalonate sapogenin diatom
and .
This article isThis article by protected copyright. All rights reserved. biotechnology sectors ran wide a have triterpenes conjugated glycosylation 2013) al. et Moses 2015; al. et Kvasnica 2011; oxidation sapogenins termed are triterpenoids Triterpenoids (C20) classification tocopherols are Terpenoids 2016; etal. etal.2013; Rouck 2017) Paddon flavors with A with interaction and defense that metabolites secondary of variety wide a produce Plants INTRODUCTION production been has Accepted Articleterpenoids and flavonoids lkaloids,
, ebr o al he cass alre classes three all of members
while triterpenoids consistof six ,
fragrances
(emtyain n the and (de)methylation , artificially
of more complex high of more complex . , ,
T
e.g. which converts sapogenin backbones into backbones sapogenin converts which he number of isoprene units that are used in their synthesis their in used are that units isoprene of number he osiue wd ad ies cas f ln ntrl products. natural plant of class diverse and wide a constitute
a
diterpenoids consist of four isoprene units and have twenty carbon atoms atoms carbon twenty have and units isoprene four of consist diterpenoids
and tutrly ies group diverse structurally produced in a microalga, a in produced (Thimmappa et (Thimmappa al. 2014; Augustin etal. 2011) colorants the -
environment value, metabolites Mss t l 2013; al. et (Moses
diin f irgn r ufr atoms sulfur or nitrogen of addition are the major classes of plant secondary metabolites metabolites secondary plant of classes major the are that isoprenoid ge of applications in the food, health, and industrial and health, food, the in applications of ge d big xlie as exploited being ady
. fe undergo often
demonstrating
, . (Moses & Pollier 2013; Sawai & Saito 2011) Saito & Sawai 2013; Pollier & (Moses
One of the most common modifications is is modifications common most the of One which which
units and have thirty carbonhave thirty units andatoms
in micro Bourgaud et al. 2001; Bansal et al. al. et Bansal 2001; al. et Bourgaud
includes amph
hmcl modification chemical
the feasibility of the of feasibility the algae are involved in development, development, in involved are ipath
carotenoids .
high . ic
saponi - au therapeutics, value
determines their determines (Augustin et al. al. et (Augustin n , s.
sterols and and sterols Simple and and Simple
photo sc as such s
( C Basic Basic - 30) bio . - .
This article isThis article by protected copyright. All rights reserved. with has hosts microbial relevant industrially in compounds 2015) al. et Huang product end the enzymes native of presence the a on grown be cannot or procedure unfeasible often i and hosts natural their the Although (Cichewicz & Kouzi 2004;Dragetal. et 2009;al. 2011) Kommera of treatment the for potential shown have compounds these of stud this In amyrin triterpenoid glycosylations catalyze which (CYP) (OSC) biosynthesis triterpenoid The Acceptedncreasing Article eeat biosynthetic relevant
in s , which mediate oxidations mediate which , , which construct the basic triterpenoid basic the construct which , ignificant efforts ignificant
, complex mixtures of related compounds related of mixtures complex -
amyrin, y h yield the ,
in plants in re is substantial is re scaffolds we focus we , ,
saponins derive from intermediates of the phytosterol pathway and pathway phytosterol the of intermediates from derive saponins
as seen in seen as dammarenediol and . s most as
is often is Therefore of ed involves being
ae en identified; been have agt t target
on lupeol on
nye in enzymes cost Nicotiana benthaniama Nicotiana medicinal that
undertaken , industrial
the investigation of biotechnological routes to produce these produce to routes biotechnological of investigation the a minimum of three of minimum a Swi Sio 2011) Saito & (Sawai ly, ri
, can
terpenoid and uridine diphosphate uridine and
, as the as betulin and betulinic acid (BA) acid betulinic and betulin
suitable make make
to lupeo
plants interest
se compounds se to isolate the isolate to te pat species plant other s undesirable undesirable
l
n h plant the in (Moses et al. 2015)(Moses etal. commercial scale. commercial scaffolds, do not have an established transformation transformation established an have not do oe f h bs characterized best the of some in , mak
triterpenoids, large triterpenoids,
enzyme and ing .
key enzymes involved in these plant these in involved enzymes key
modifications to modifications n aue mr ta 10 distinct 100 than more nature, In
are are cytochrome P450 monooxygenases P450 cytochrome become an active fi active an become Oryza sativa Oryza
ta ntrly rdc them, produce naturally that s their isolation their
dependent glycosyltransferases, dependent
usually classes
s fe comp often is . Additionally .
triterpenoids. Derivatives Derivatives triterpenoids. HIV : oxidosqualene : present in low amount low in present
(Khakimov et al. 2015; al. et (Khakimov - scale extraction from from extraction scale
n cran cancers certain and difficult. intermediates or intermediates
eld of research of eld the transfer of transfer the licated
include Moreover, Moreover,
cyclases
y the by typical
to to is s - -
This article isThis article by protected copyright. All rights reserved. Accepted al. 2009; etal.2016) Arendt heterologousterpenoid for exploited successfully with breakthroughs important seen have biology synthetic in Advances feeds aquaculture and animal oils pigments, as 2015) al. biomass et algal Moreover, Gimpel 2016; al. et Ruiz 2012; al. et Work 2013; al. et Wijffels 2013; al. et production of impact environmental sunlight utilize Article2015) yeasts and bacteria are production terpenoid for hosts heterologous popular most the Although al.2017)(Pateraki et et al.2015; Rouck as such 2016) prokaryotes in rare but eukaryotes all in present is that pathway biosynthesis as triterpenoids of production the for preferable seem specific
oe htsnhtc hosts photosynthetic some , Ad .
(Møller et al. 2008;(Mølleral. et there has been increasing interest in interest increasing been has there isnhtc pathways biosynthetic shrci coli Escherichia ditionally n CO and
and ,
cytochrome P450 enzymes enzymes P450 cytochrome
omega 3 fatty acids fatty 3 omega 2
n etnie niern is engineering extensive and o obtain to c
Zhou et al. 2016; Leavell et al. 2016; Kirby et al. 2008; Moses et al. 2016;et al. Leavellal. Moseset al.Kirby Zhou 2016;2008; et an .
be industries . n general In a ,
including useful source of source useful energy
(Davies et al. 2015; Kyle J. Lauersen et al. 2016; Work Work 2016; al. et Lauersen J. Kyle 2015; al. et (Davies .
.
,
which can find can which , and
photosynthetic uaytc hosts eukaryotic ynbcei ad os ise cultures tissue moss and cyanobacteria carbon generally generally
production
a variety of value of variety a
fe rqie t oti hg titers high obtain to required often o growth for they can tap into the existing sterol sterol existing the into tap can they
application in application o o fnto wl in well function not do microbial microbial
(Davies et al. 2014; Anterola et Anterola 2014;et al. (Davies like
Saccharomyces Saccharomyces , in terpenoid engineering engineering terpenoid in
hrb minimizing thereby - add platforms, as they as platforms, the ed food, cosmetics, cosmetics, food, molecules such such molecules
(Wei et al. al. et (Wei cerevisiae bacteria ,
being
can the the .
This article isThis article by protected copyright. All rights reserved. in produced limited are modification genetic stably and photoautotrophically diatom, Phaeodactylum genomes nuclear unicellu 2016) al. et Hamilton 2001; Ohno & Wikfors 1998; Nrel 2004; aquaculture for production existing and content lipid high their engineering metabolic and biotechnology for attractive increasingly processes industrial in deployment systems algal other processes, physiological of elucidation the for organism model good a be to proven 2016) al. et Lauersen J Kyle 2016; al. et Erpel 2016; al. et (Shin those identify and marker resistance the T genes these genes heterologous of expression 2016) al. 1962) Calvin of aspects and photosynthesis reinhardtii M herefore,
Accepted Articleas such algae green on performed been has research microalgal of ost
a age ht r gntcly tractable genetically are that algae lar . P.
However extensive screening extensive as they have been historically historically been have they as . are prone are P. tricornutum P. Recently a sesquiterpenoid (C15) was produced in this alga this in produced was (C15) sesquiterpenoid a Recently tricornutum are
tricornutum
better suited for suited better nld the include scesu egneig f re algae green of engineering successful ,
to gene silencing mechanisms silencing gene to ,
has already already has
include antibodies and biodegradable plastics biodegradable and antibodies include Vee e a. 02 Bwe e a. 08 At t l 1996) al. et Apt 2008; al. et Bowler 2012; al. et (Vieler
E physiology needs to be to needs ustigmatophyte express express
that . both .
C n eet years recent In
mecal itrsig rdcs ht ae been have that products interesting ommercially have been have fu express the
such as circadian clocks circadian as such hc epes h transgene the express which show nctional undertaken adopted n Nannochloropsis
integrated and industrial potential as it can grow grow can it as potential industrial
enzymes
(Cerutti et al. 1997; Rasala et al. 2014) al. et Rasala 1997; al. et (Cerutti ion of heterologous pathways heterologous of ion ,
have
h heterokont the
s oe sses t systems model as to weed out lines that only express express only that lines out weed to
into publically available available publically novs overcoming involves , although although ,
(P. Kroth 2007; Pulz & Gross & Pulz 2007; Kroth (P. the nuclear the . Two examples of brown brown of examples Two .
. oceanica (Noordally & Millar 2015; Millar & (Noordally
While While
applications
algae molecular tools for for tools molecular o ufcet levels sufficient to (Kyle J Lauersen et Lauersen J (Kyle . reinhardtii C.
Chlamydomonas and the diatom diatom the and (Franziska et al. al. et (Franziska genome
o understand understand o have become become have
because of because sequenced
the
and for for and . Often . . poor poor The The has .
This article isThis article by protected copyright. All rights reserved. the of genomes localize generally enzymes These oxidosqualene the of members by cyclized is which oxidosqualene, Triterpenes, Expression of RESULTS plant secondary ( triterpenoid for betulin and also enzymes products family oxidation C28 partial CYP716A produce the of Most BA. to P450 cytochrome family CYP716A a by position C28 the at oxidized subsequently is which lupeol, produces OSC synthase lupeol reductase. its with along P450 heterologous in betulin) and (lupeol triterpenoids plant two of production efficient cytosolic the production. non C inperformed thishasbeenon organism 2011) al. et Hempel 2011; Accepted Article the through exclusively (IPP) diphosphate isopentenyl form algae green and yanobacteria - moss eaoae aha (E) IP s h ky rcro fr trl n ohr isoprenoid other and sterol for precursor key the is IPP (MEP). pathway mevalonate
and DISCUSSION
hsoirla patens Physcomitrella aldehyde Conversely, b Conversely, the mevalonate ( mevalonate uh as such two xrsin f ln enzyme plant of expression metabolites.
C micro
30) production and opens and production 30) l upeol synthasesupeol in . This is the first report of genetic engineering of a eukaryotic microalgaeukaryotic of a engineering of genetic report first the is This
algae algae trl, r snhszd i te 30 the via synthesized are sterols, oth
. MVA
To the best of our knowledge, our of best the To
Chlamydomonas higher plants higher In the In )
and to contain plant betulinic acid (BA) acid betulinic plant the ER and ER the the chloroplastic the P. , the the
tricornu which
s single a and : carotenoid pathway carotenoid
the way the an
reinhardtii, ,
diatoms
in the case of lupeol oxidation lupeol of case the in tum contain oxidosqualene
oxidosqualene
results in results for photo for
MEP
such as such a hedcyu tricornutum Phaeodactylum membrane the only terpenoid engineering terpenoid only the
pathway. pathway. biosynthetic pathway biosynthetic - - l
bioproduction of high of bioproduction abn intermediate carbon upeol accumulation P. tricornutum P.
cycl (Eilers et al. 2016) al. et (Eilers
yls (S) ee for gene (OSC) cyclase s ad a and ase
P. tricornutum P. yls (S) family. (OSC) cyclase binding domain binding Here , w ,
contain both contain e report the the report e , cytochrome
are betulin are . (Fig
.
by the by , -
value . .
2,3 1) a 1) and and The The -
This article isThis article by protected copyright. All rights reserved. lupeol synthaseandotherunknowntriterpenesynthases evolution plant higher previously LUS lanceolate Bupleurum beta beta with analysis for enzyme OSC an select To (Segura et al. 2000; Salmonetal.2016) of production T etCzarnotta al.2017;& etal. Lewandowski2016; Lang Zhou 2016) yeasts in production acid betulinic and lupeol for used characterized been has LUP1 ⁄ At1g78970 skeletons triterpene contains genome genomes 2,3 substrate single the from derived diversity chemical of tetracyclic h
Accepted Articlee Arabidopsis thaliana Arabidopsis - mrn synthase amyrin of - contain
amyrin synthases amyrin sterol biosynthesis sterol that selected lupeol and lupeol either two branches two either from thirteen plant OSCs plant (Vincken et al. 2007; al. et (Vincken
or or nine to nine A) wie all while BAS),
lupeol synthase lupeol the related triterpenoid, 3β, 20 3β, triterpenoid, related the , that
Lotus japonicus Lotus OSC such as the as such . H . introduction , AtLUS , sixteen with known functions known with ee family gene plants igher of lupeol synthase genes have been generated been have genes synthase lupeol of
represents . OSC
Artemisia
Xue et al. 2012) al. et Xue (from now on now (from uel synthase lupeol
(Herrera et al. 1998) al. et (Herrera
in OSC3, genes ,
enzymes in contrast in P. tricornutum P.
Olea europaea Olea a
e.g. the e.g. annua n evolutionary
(Fig. S2) (Fig. n cn rdc a ies ary of array diverse a produce can and
, designated designated
(Shibuya etal.1999)
- can contain a remarkable amount remarkable a contain can
. OSC2 dihydroxylupane
model plant model (Husselstein s A
, - mong
oxidosqualen (LUS) (LUS) we ;
, successfully expressed and expressed successfully , i and nterestingly
LUS. performed .
transition state transition these,
the lse sprtl i separately cluster as It has It – AtLUS) catalyzes the the catalyzes AtLUS) Arabidopsis thaliana Arabidopsis Muller et al. 2001; 2001; al. et Muller Catharantus roseus Catharantus
a lupeol synthase, lupeol a . yial plant Typically e. ,
as a by a as , been
a phylogen a . AtLUS clusters AtLUS
suggested
between between - product product during et .e. .e. ic ic
This article isThis article by protected copyright. All rights reserved. to bescreenedon forfluorescence primarytransformant plates which of one transgene, of the copies to employed production with favorably a using identified and expressed functionally each for lines screened the of 30% production GC initial the on Based ( lines to subjected transfer t Single ( genome of sequences coding prevalent in functional be to shown been have enzymes Both expressed To FCPA/LHCF1
Accepted Article et h faiiiy of feasibility the test Fig. red asomn lines ransformant ne te oto o te tog uoati clrpyl a/ chlorophyll fucoxanthin strong the of control the under
2) in literature in t ,
of wo
to with the identification supported by the MS electronimpact MS the by supportedidentification the with
) hr more where GC select select
lupeol ( lupeol
promoter liquid medium medium liquid representativ - the MS analysis MS eaiey easy relatively positive lines positive Supplementary t Supplementary
. reinhardtii C. (Sawai et al. 2006; Zhou et al. 2016; Czarnotta et al. 2017) al. et Czarnotta 2016; al. et Zhou 2006; al. et (Sawai AtLUS along , - MS P. tricornutum tricornutum P. intricate that grown on selective solid medium medium solid selective on grown
OSC e cenn w selected we screening and l and and
n grown and with P. tricornutum P. ,
LjLUS includ upeol was detected was upeol n low and s
:
etr desig vector ltom sd for used platform AtLUS ecn eitne ee akr ( marker gene resistance zeocin was able 1). On av On 1). able construct
ing ee nertd no the into integrated were s a as fused with a reporte a with fused
ni ery ttoay phase stationary early until the preparation of a construct a of preparation the or throughput
ou japonicus Lotus uel production lupeol lines expressing heterologous genes can be can genes heterologous expressing lines .
n hs established This
S. erage, we were able to detect lupeol in in lupeol detect to able were we erage, and th cerevisiae in ose
screening
cenn mtos ed o be to need methods screening both sesquiterpene
lines with the highest relative relative highest the with lines r
(Kyle JLauersen etal. 2016) LjLUS S3 ( OSC3 , gene (YFP)to gene
with AtLUS being the most most the being AtLUS with zoi antibiotic) (zeocin
platform
method . tricornutum P. that the enzymes are are enzymes the that
and fr ble c
om . spectra (Fig. S3A (Fig. spectra
idn poen A protein binding r Thirty patchoulol ) AtLUS
. ,
harboring S1 A n B) and A (SF1, now on on now This
e ectopically we allow colonies colonies allow
.
lines were were lines expressing expressing T compares compares he he
nuclear , LjLUS)
nati three (C15) we - ve R re ) . . .
This article isThis article by protected copyright. All rights reserved. (Fig. grouped together a by supported as different significantly 0.01 < p 7.17, = F(7,20) expression in difference significant a was there that showed lines selected the in expression lines q between genome nuclear the into transgene t Nuclear number ofintegrated high with correlates productivity lupeol High tricornutum (Fig synthase cycloartenol was oxidosqualene, 3 similarity structural or sequence upon based identified been has enzyme epoxidase squalene canonical no Interestingly, microalga. first therefore, conditions culturing dihydroxylupane line transformant the In AcceptedRT Article -
PCR on the on PCR ucsfl demonstration successful ht hwd h high the showed that
higher lupeol production and production lupeol higher asomto eet in events ransformation
focused our attention on line LjLUS line on attention our focused
, as the introduced the introduced three
by - , product
LjLUS
expression expression hl LLS xrsig ie produce lines expressing LjLUS while 3 predicted predicted . The line with the best lupeol accumulation, namely LjLUS namely accumulation, lupeol best the with line The . s A
) expressing . .
1). 1).
est lines otn rdcd at produced often ,
lupeol
This result result This uel accumulation. lupeol of
that cassettes o be to pentacyclic . tricornutum P. AtLUS
I odr o test to order In . synthases showed the lowest the showed relative
post hoc post present affirm ,
we were able to identify lupeol and the 3β, the and lupeol identify to able were we (Fabris et al. 2014) al. et (Fabris -
LjLUS 25. require rtreod production triterpenoid trans s
Tukey test ( test Tukey
as h peec o 2 of presence the To the best of our knowledge our of best the To
similar ed o adm insertions random to lead
expression
gene the substrate of the well annotated annotated well the of substrate the oe a AOA nlss f LjLUS of analysis ANOVA way one A
this precursor to
whether whether lupeol accumulation and accumulation lupeol
expression levels, we performed we levels, expression titers α d
, although its product, the 2 the product, its although , = 0.01) while the other lines other the while 0.01) =
but , just
the but also dependent on on dependent also but - is independent is oxidosqualene 3 re re uel (Fig lupeol
synthesize synthesize is in
in any correlation correlation any a P. tricornutum P. n engineered engineered n
, of a given given a of
. this is the is this
the three the - lupeol. 2) 25, was was 25, on .
in We, We,
the 20 P. - -
This article isThis article by protected copyright. All rights reserved. effects mRNA correlation betwee ( 3B figure in indicated are other each from different significantly groups and lines the of comparison the for performed were comparisons hoc were there that but integrated of number the between difference no was there that confirmed analysis This transgene. integrated the and lines individual the of effects events integration (Fig abundance: similar have to found determined the of number copy UBQ amplifying by q integration genome integration T investigate o Accepted ArticleRT . -
PCR for PCR Phatr3_J 3B) expression rather thanthe mN epeso levels expression mRNA n This .
score of score Bt transgene Both .
r u to due or the three three the
regions within regions
28620
is whether consistent and lupeol accumulation lupeol and (Falciatore et (Falciatore
) - significant differences between the lines the between differences significant events , 0.03, t = t 0.03,
LjLUS for
low and low absolute rngn cp nme rslig rm ige or single from resulting number copy transgene
which the copy number is assumed to be t be to assumed is number copy the which mRNA
, oig sequence coding
the s two ih ht a been has what with 0.07
ee rsn o a ige rnfrain etr and vector transformation single a on present were three number of number approximate transgene copy transgene approximate al. 1999) al.
xrsin level expression wild different , p > 0.9 > p , two to three three to two and
high the
. lupeol
) integrated transgene A . ubr of number
are n Th
n te ecn eitne marker resistance zeocin the and
ANOVA was conducted to compare the main main the compare to conducted was ANOVA ese results ese - transgene copies transgene
likely type genomic type producing
s rvosy reported previously r mil determi mainly are due to due integrated α
00) Tee was There =0.01).
suggest that suggest strains
ble position (p<0.001, F(6,29)=26.49) (p<0.001, number was number loci r copies
for each for and .
LjLUS ( Results were were Results RP3a wo wo al LjLUS .
variations
in a in
(site of integration) integration) of (site
for cassettes Phatr3_J13566 e by ned transformant line transformant
multiple
quantified using using quantified diploid cell diploid copies
. tricornutum P. no correlation correlation no normalized ( ble in relative in
(Pearson (Pearson locus of of locus
(p=0.29) (p=0.29) nuclear nuclear r ) . Post Post . were were were . The The .
and
This article isThis article by protected copyright. All rights reserved. consistent of expression the between difference ( significant was time in difference ob productivity lupeol the with line transcripts phase. stationary to exponential from transitioned cells the which during days five for also We (Fig retained minimal was stage this No reached produced lupeol of amount brassicasterol metabolism in arrows the by indicated during lupeol and sterol) main (the brassicasterol day at biomass experiment performed during productivity lupeol the monitor to order In cell and dependent phase growth is lupeol of Production
Accepted Article
brassicasterol was observed was brassicasterol .
4 B ).
after twodaysofgrowth tracked within the cell the within
with the with showed t h ery tg o ter growth their of stage early the at e cells per s
gene expression of expression gene , , , n 5 f growth. of 5 and 2, 1, 0,
FCPA/LHCF1 aiu epeso level expression maximum s ,
in . in lupeol is able to able is lupeol I apas that appears t an the
F per cell (Fig cell per igure Algem L
in either the LjLUS or WT medi WT or LjLUS the either in j promoter activity, which activity, promoter . LUS
.
4 ® LjLUS and (Fig tained - )
25 , labscale labscale () 63, p=0.005 6.32, F(3)= LjLUS suggesting that cells are actively engaged in sterol sterol in engaged actively are cells that suggesting
.
line 4
actively or passively translocate passively or actively and while native sterols such as brassicasterol are are brassicasterol as such sterols native while A)
. C and and
when photobioreactor, collecting and analyzing the the analyzing and collecting photobioreactor, The m The max reached ells the s .
4 W . uig mid during ble C zeocin resistance zeocin ). oprd o WT, to compared aximum r could e
n NV analysis ANOVA An F1=., p=0.41). (F(1)=0.7, mid reduces reduces
h different the peaks peaks bt hr ws o significant no was there but ) - exponent -
xoeta got pae in phase, growth exponential yield of yield also a imum b during exponential growth. growth. exponential during , suggesting cellular lysis at lysis cellular suggesting , rassicasterol observe observe confirming protein confirming
a got phase growth ial
lupeol productivity of both both of productivity growth consistent
This
hwd ht the that showed into the medium medium the into rdcin of reduction a in the cell the in
result is also also is result levels in the the in levels phases
with
we , Both Both
was ble the ( as as r
This article isThis article by protected copyright. All rights reserved. a and process overall value multiple of extraction platform the to value is and addition, In is obtainwesystem microalgal titers supply cofactor elevated and pathway mevalonate the into flux increased of titers acid optimization achieved comparison For (Kyle J sesquiterpene mg mg·L 0.1 of (C30) yield Strain promoter the While
Accepted concentrationshigher thantheinitial Article / L
reached yield LjLUS Lauersen etal.2016) ih in rich proposed
in could potentially enhance could potentiallyenhance LjLUS activity. FCPA/LHCF1
- a f lupeol of 25 was selected as the best produci best the as selected was 25 hs first this rounds and rounds
S. cerevisiae S. . mg/L 0.1 patchoulol fucoxanthin 21 gg r biomass dry mg/g 12.1 economics. :
the irfnr approach. biorefinery -
generation generation
- highest
1 civd in achieved /OD promoter is widely is promoter .
over f F ed
(C15), grown in grown (C15), rhr piiain in optimizations urther strain . and In ed - Hag t l 2012) al. et (Huang
-
batch cultivations. The cultivations. batch de molecules added two
F yield titers > 0.1 mg/g dry biomass with biomass dry mg/g 0.1 titers> igure long (Czarnotta et al. 2017) al. et (Czarnotta
. tricornutum P. days of culturing. of days . reinhardtii C. - o btlnc cd production acid betulinic for
chain chain 5
reported for reported
we report the extraction procedure of triterpenes triterpenes of procedure extraction the report we L ad hn 21; i n Zag 2015). Zhang and Li 2014; Zhang and (Li
comparable used, Sources for this analysis can be found in in found be can analysis this for Sources poly
ng line. LjLUS line. ng r epce to expected are -
, nauae fty cd ( acids fatty unsaturated strain a stronger and growth phase independent phase growth and stronger a
n with and
nierd o te rdcin of production the for engineered
Th the first eeis and genetics is
conditions a b grown be can .
primary -
reported yield reported generation strain generation T his his ute gntc improvements genetic further - 25 reached a reached 25 strain is the result of multiple multiple of result the is strain
yeast strains
substantially , no
over a seven day period period day seven a over utr conditions culture (
prior 182 photoautotrophically is
similar mg optimization
lc showed maximum maximum UA) add PUFAs), , .
·L
betulinic acid acid betulinic - im 1
) to the 0.35 0.35 the to
prove ha betulinic betulinic
s n o In
lupeol , been ,
and and this this the the the ing ur ur ,
This article isThis article by protected copyright. All rights reserved. biosynthesis the 2005) Goodman & Hsieh 2004; al. et (Suzuki deleteriou severely and distinct in results pathway (Rodríguez inter IPP minimal or no be to localized chloroplast ER. the in made is pyrophosphate isopentenyl cellular In transcripts the affects production Triterpenoid molecules value multiple of extraction and conditions, culture and genetics in optimizations sugars) and (protein biomass phytosterols 2022) co allow procedure purification microalgae 4. table supplementary - Accepted eukaryotic Article rdcs include products chloroplast , carotenoids carotenoids ,
compartments. Both carotenoids and sterols are produced are sterols and carotenoids Both compartments. , -
are expected the toincrease competitiveness of platforms microalgal in in ocpin 2006) Concepción and
and photosynthetic pigments. and photosynthetic photosynthetic organisms different terpenoid classes are produced in different different producedin are classes organisms terpenoid different photosynthetic uh as such the
localized estimated LjLUS Higher plants and plants Higher
MEP pathway MEP eicosapentaenoic acid acid eicosapentaenoic uh as such brassicasterol - 25 line transformant The MEP pathway MEP
(IPP) s ot ae equal are costs extraction
can still be still can . the separation of other of separation the change Indeed, fucoxanthin ,
but the former is made in the chloroplast while the latter the while chloroplast the in made is former the but
(Fig. 1). Whil 1). (Fig.
expression levels of native sterol metabolism sterol native of levels expression P. tricornutum P. $25 million ($62.5
between the cytosol and chloroplast and cytosol the between
disruption of key genes in either the MVA or MEP MEP or MVA the either in genes key of disruption procedure
which supplies which used
.
. ( crety 9 million $95 (currently G market as a lo a as Refining the microalgal biomass from this this from biomass microalgal the Refining reen algae such as such algae reen e both pathways produce IPP, there appears appears there IPP, produce pathways both e
for
contain both the cytosolic MVA and MVA cytosolic the both contain w value w by market global phenotypes s valuable
est triterpene
IPP for both both for IPP
imated to reach $3.79 billion by by billion $3.79 reach to imated animal food animal molecule
is the same for yeast and yeast for same the is C
from .
in reinhardtii
the lbl market global Arabid 00. The 2020). s. the same precursor precursor same the
.
ER Therefore, further further Therefore, Examples of these these of Examples
in higher plants higher in localized sterol sterol localized o ss thaliana psis
only contai only .
defatted defatted enzyme - added added ) and and )
the n
This article isThis article by protected copyright. All rights reserved. increased ( the and Table (Supplementary th LjLUS of Expression and line overexpression LjLUS correla the and type wild the both for true holds This five. day phase exponential immediately was days five Samples whether pathway MEP localized chloroplast enzymes biosynthetic sterol of set selected a of expression or biosynthesis sterol affect non a of introduction The Phatr3_EG02290
Accepted model ANOVA measures repeated a using tested was genes investigated e Article - 25 line compared to the wild type wild the to compared line 25 tes wellwiththelupeol andbrassicasterol levelsmeasured (
ectopic for of culturing. of culturing.
only slightly spney dpopae isomerase diphosphate isopentenyl RNA extraction were taken were extraction RNA some of the of some LjLUS
) are are ) apparent that expression of sterol biosynthesis enzymes is higher during higher is enzymes biosynthesis sterol of expression that apparent and The algae The .
influences native sterolbiosynthesis expression.influences
consistently upregulated consistently
5 nearly all investigated genes genes investigated all nearly ). -
native OSC such as the LjLUS in LjLUS the as such OSC native mevalonate pathway mevalonate ee cdn fr k for coding Genes h IP rdcn pathways. producing IPP the
started
n ctslc V pathway, MVA cytosolic and
enter from cultures of cultures from
as shown in figure 6 figure in shown as ing y nye sc a HG ( HMGR as such enzymes ey , while ,
stationary phase by the by phase stationary encoding genes genes encoding
-
reached their expression their reached s
uln synthase qualene HMG
wild type and LjLUS and type wild P. tricornutum P. - CoA synthase ( synthase CoA , including enzymes enzymes including , . T
The statistical significance of significance statistical The herefore increased markedly in the in markedly increased F
ig by qPCR to determine determine to qPCR by .
4A
f we - ifth
B
could potentially potentially could ). fusion enzyme enzyme fusion
Phatr3_J16649 Phatr3_J16649
-
day ( day measured the the measured 25 line during during line 25
minimum on on minimum Fig. from the the from
S 4).
It It ) )
This article isThis article by protected copyright. All rights reserved. Andr 2011; CYP716A 2013) Bak & (Hamberger a by In o I 2000)Rohmer the in production triterpenoid and biosynthesis sterol for IPP of source major the is pathway MVA in any if biosynthesis, sterol in role its and lines transgenic in pattern upregulation consistent pathway MEP ( ( m a includes oxidosqualene. Phatr3_J46726 Surprisingly, attempt an in synthesis brassicasterol in involved genes of upregulation LjLUS the in lower are brassicasterol of levels the Since ntroducing Phatr3_J51757 Phatr3_J48864 xidation
P. tricornutumP. P. tricornutum P. Accepted(e.g. species plant higher several Article P. tricornutum tricornutum P.
yohoe P450 cytochrome
family family of lupeol e a. 06 Mss t l 2014) al. et Moses 2016; al. et e a no upregulation upregulation no . transcripts
CYP716A12
, C4 trl euts ( reductase sterol ) C24 a ), However, enzyme transcripts downstream of downstream transcripts enzyme However,
to be responsible for this modification this for responsible be to uin ee tog i cmee wt the with competes it though even fusion) , remains unclear. remains , . with all of these enzymes showing a similar similar a showing enzymes these of all with ethylsterol This is in agreement with earlier studies, which showed that terpenoids in in terpenoids that showed which studies, earlier with agreement in is This
to cytosol and chloroplast do not have the the have not do chloroplast and cytosol
betulinic acid - dependent
IspE .
cytochrome cytochrome eety a numb a Recently,
( o Phatr3_EG02383 yeae ( xygenase was
Overall these results suggest results these Overall platyphylla Betula
monooxygenase seen seen P450 for Phatr3_J10852 r of er
and and . ) and ) the
ae o te ieaue e eetd the selected we literature the on Based Phatr3_J48260 a studie PtOSC ) ISPD2 P450 (CYP) ,
- lupeol (Carelli et al. 2011; Fukushima et al. et Fukushima 2011; al. et (Carelli 25 line 25
s -
NADPH reductase reductase NADPH ), a sterol dehydrogenase dehydrogenase sterol a ), ( have o om euii acid betulinic form to rncit ( transcript Phatr3_J21829 PtOSC ae ore f IPP of source same is gene gene ,
that, as in higher in as that, we expected to see marked marked see to expected we oxidized reported ad 2 reductase C22 and ) jU ezm fr 2,3 for enzyme LjLUS
expression pattern. expression show Phatr3_EG02293
at the C28 position position C28 the at upregulation.
) nye o the of enzymes to compensate. compensate. to
did not show a a show not did leads to the the to leads plants, the plants, (Cvejić & & (Cvejić
(Fig
This This . T
he he 1) + -
This article isThis article by protected copyright. All rights reserved. Accepted was fused order CYP716A12 work this In CYP enzymes catalytic increased 2004) al. et Zangar 2010; Møller & Jensen species oxygen reactive of release host same the Articlecerevisiae terms functional in complement partially least at to species different from CPRs of ability the by demonstrated as conserved, well are domains the to NAD(P)H from electrons shuttle which Cytochrome P450 workintandemwithspecific enzymes oxidase activities. with enzyme multifunctional assays truncatula Medicago ,
to ensure an adequate supply of electrons to the to electrons of supply adequate an ensure to
to n es cnimd that confirmed yeast in
increase catalytic efficiency of the CYP the of efficiency catalytic increase
or and to its
(
w , MtCYP716A12 a (Schückel et al.2012;(Schückel Leonardetal. etal. 2006;2007) Munro P. tricornutum P. related species related native tse te expression the tested e
efficiency
CYP716A12
MtCPR co ) ,
has co contain
β for - - - expressed expressed
mrn 28 amyrin enzyme. We enzyme. and CPR for expression in expression for CPR and been obtained been
maximum (Jennewein et al. 2005; Kim et al. 20 al. et Kim 2005; al. et (Jennewein the a native CPR, native eiao truncatula Medicago in - along . oxidase,
activity T therefore . tricornutum P. cytochrome P450 cytochrome his
– by fusing by
CPR ih the with euto se cn e ae limiting rate be can step reduction CY when
α P enzymes P system - employed
mrn 28 amyrin . cytochrome
CYP
lhuh uayts uh as such eukaryotes Although one or more CPRs more or one P. tricornutum. P.
. truncatula M. introduced
, and of the
.
CYP716A12 usually require usually The CPR The t potentially two approaches: he - MtCYP716A12 xds ad uel 28 lupeol and oxidase
P450 reductases (CPR)
Medicago in 09; Kim et al. 2005; 2005; al. et Kim 09; to . –
In
P (MtCPR). CPR P450 interacting interacting P450
- a minimizing the the minimizing - directly enzyme house a house heterologous
a truncatula
sequence sequence CPR from CPR
to the to ctivity
s a is and
In S. S. - ,
This article isThis article by protected copyright. All rights reserved. 3 native the Acceptedλ linker coding DNA of stretch short additional an by spaced were sequences coding or insertion membrane domain transmembrane with without MtCYP716A12 roseus the expressed follow iterative the In three Articleof genome the into integrated were MtCPR the For 2. 1.
the
pla ing
in MtCYP716A12 best the and production construct iterative An and transformed co A ( co GSTSSGSG), to GSTSSGSG), s
exclusion of the the of exclusion E. mids a strategy strategy a -
transformation Mt c - oli srcue f the of structure D rnfrain prah wherein approach transformation CPR
G (Supplemental (Supplemental
lycine (Leonard & Koffas 2007; Leonard et al. 2 al. et Leonard 2007; Koffas & (Leonard
approach, and
coding sequences
approach with nprd y h work the by inspired -
CPR the max
the prevent the fo the prevent association
( Δ71 the
ble and the and
is 7 first approac P450 isoflavone synthase (IFS) fused to a CPR from CPR a to fused (IFS) synthase isoflavone P450 stop codon was fused in frame to frame in fused was codon stop we
CPR) r , LjLUS figure 1)
gene where
1 fused tan a frhr rnfre with transformed further was strain
. nourseothricin mn acids, amino , , the h,
The membrane anchor anchor membrane The
two + which could which . marker
zeocin rmation of secondary structures secondary of rmation . . tricornutum P.
the enzymes native of The . P. tricornutum P. . truncatula M.
eitne gen resistance enr ad Koffas and Leonard
hc are which compromis
ee encoding genes
resistance . resulting
h r The . tricornutum P.
a frt rnfre wt an with transformed first was
006) esulting final coding sequence, sequence, coding final esulting
in a single transformation single a in was predicted predicted
ie wr sree for screened were lines e CYP e selection marker
akr ( marker . the enzyme the
the T 716A12 deleted he full coding sequence of of sequence coding full he wee hy functionally they where , LjLUS, MtCYP716A12 and and MtCYP716A12 LjLUS,
MtCPR coding sequence sequence coding MtCPR
to , a simultaneously was ble
which
fus a to avoid unwanted unwanted avoid to and correspond to the the to correspond r
) activity MtCYP7 ,
(nat ed P enzyme CPR could for a flexible flexible a for C atharantus version of of version r . )
. T
he two two he disrupt 16A12, 16A12, , using using , lupeol lupeol
OSC ,
This article isThis article by protected copyright. All rights reserved. reinhardtii currently availabl compare transgenes co single a in plasmids different In brassicasterol, making the quantificationbyGC 2011) al. expressing yeast recombinant in identified product main the also showed lines fusion two and line transformation S stan known of those with patterns fragmentation impact electron the comparing intermediate first in resulted strategies and combinations All phase medium liquid into transferred t the approaches, both For 2). promoter MtCYP716A12 3
E Acceptedthe Article
- I ). .
co
O fr selection for e - d f the the f . transformation approach transformation . This vector This .
vial for available to the step the to As shown in in shown As . where thefrequencies where for thesuccessful expression transgene ofasingle hs prah a mil amd to aimed mainly was approach This - λ thirty - Δ71 in
CPR
needn lines independent - euii ai production acid betulinic wise transformation, wise was used was , , was cloned was , Fig . tricornutum P. with ransformant line ransformant .
7 te ek f eui i vr lw compar low very is betulin of peak the , only only
and , to transform to , we showed that showed we , - two rnfrain event transformation under the control the under metabolites metabolites
that ( el characterized well ble and s transformant
r
were were the best lupeol producing line: LjLUS line: producing lupeol best the were were - and analysis
to clear (Fig reduce side
nlzd y GC by analyzed grown on selective on grown P. tricornutum P. nat cend e transformation per screened .
peak 7
- r challenging )
). step t step .
the of
ie n cs o transformation of cost and time ek dniy a cnimd by confirmed was identity Peak s This compares compares This lines that produced betulin, the the betulin, produced that lines
for n functionally and
he niitc selection antibiotic betulin. This intermediate is is intermediate This betulin. P. tricornutum P.
this enzyme this limited
. is able to integrate to able is
- MS
solid medium solid
n al stationary early in resistance marker resistance ed favorably
to lupeol and and lupeol to (Fukushima et (Fukushima
express FCPA/LHCF1 dards ( dards ,
, such as , suchas - marker n co one 25
,
to three
then then (Fig F the ig C. C. s s - . . ,
This article isThis article by protected copyright. All rights reserved. well toleratedatthelevels produced. reductase and the Fig lines producing lines transformant a have could T analysisGrowth andscaling performance up process tricornutum using production acid betulinic and betulin of efficiency limited with modification. engineering the In without screening Lauersenextensive asperformedinthe2016by study etal. 2013) al. et Rasala mutagen of usage the or marker resistance antibiotic an with linkage translation by obtained be only can expression strong luciferase, or GFP e eupsn o seo ptwy nemdae and intermediates pathway sterol of repurposing he
Accepted. Article
transformant strains transformant S 4 iterative approach iterative existing and
S , makingthismicroorganismapromising s
approach 5 This allowed us allowed This molecular ,
genes did not affect not did genes n ermna efc on effect detrimental with eei engineering genetic o significant difference significant o
from the nuclear genome vary around vary genome nuclear the from T . . This would preclude a similar approach being applied in applied being approach similar a preclude would This .
the wild type thewild herefore,
o re to
. This . ilg tools. biology ,
w e demonstrated the demonstrated e -
ein the design to
suggests
e compared we economize
iz strain
d strains ed cell
taeis for strategies
growth growth s that the i the that lhuh oe effort more Although in an Algeman in
. truncatula M. were
on
Nuet t l 20; uran t l 20 al. et Fuhrmann 2009; al. et (Neupert
rwh efracs of performances growth
promoters observed and speed speed possibility of possibility ntroduction of ntroduction ,
these results these that the products of these enzymes are enzymes these of products the that ®
ystem for ystem labscale plant pathway pathway plant
n boas accumulation biomass and
CYP716A12
adm ula gnm insertion genome nuclear random between
and antibiotic markers antibiotic and 10%
photobioreactor. A photobioreactor. successfully successfully industrial biotechnology. and it has been shown that that shown been has it and is the
establish
the wild type wild the needed needed - P system CPR OSC eosiuin in reconstitution , lupeol and betulin betulin and lupeol
c
ytochrome P450 ytochrome applying applying the o mrv the improve to
C. reinhardtii C. feasibility of feasibility , for lupeol lupeol for ,
and s shown in shownin s in system in
protein protein of the the of any of of any
04; 04; P. s
This article isThis article by protected copyright. All rights reserved. algal system for commercialization. o extraction LjLUS in lupeol of and AtLUS6 to equivalent biomass dry of amount app an using trial, this from harvested biomasses LjLUS+MtCYP716A1 for days expected the ( during prior, immediately a to due likely was This in difference any show recover I inlinedensity withtheflaskshakeculture days 25 of period CO and agitation improved lighting, pH, over were cultures LjLUS productivity F n this large scale trial, scale large this n LjLUS+MtCYP716A12+MtCPR Accepted Article the ollowing oiaey 1 roximately + - 25 MtCYP716A12 following
culture at the time of harvest. Further Further harvest. of time the at culture ptimization from dry biomass dry from ptimization LjLUS LjLUS se n lpo content lupeol and growth performance growth rw i a 550 a in grown
smaller L culture. culture. L
-
- the initial harvest initial the for the first harvest, first the for 25 25
+ lines
MtCPR line, compared to AtLUS6; this result might be due to the status of of status the to due be might result this AtLUS6; to compared line, despite displaying expected growth kinetics, growth kinetics, expected despitedisplaying n unidentified n behavior - scale trials in Algem in trials scale , as expected from the growth fitness, we extracted lower amount amount lower extracted we fitness, growth the from expected as , upeetr t Supplementary 2+MtCPR). r post or
i a eiaie f h LLS5 The LjLUS25. the of derivative a is ) n fence a in
liter
compared to the two other lines tested at the large scale large the at tested lines other two the to compared ,
of okn vlm photobioreactor volume working allowing multiple multiple allowing (fig. S6 (fig. - avsig since harvesting,
technical once
We performed a small scale extraction from the dry dry the from extraction scale small a performed We three - s
tl tblr htboeco (PBR photobioreactor tubular style will enable us to investigate the feasibility of this of feasibility the investigate to us enable will . )
. This was unexpected as growth curves did not not did curves growth as unexpected was This . able they ®
photobioreactor rngnc strains transgenic 2
performance delivery. 2 reach nlss n h sae p system up scale the on analysis
shows shows harvest
ed
n o the of one
early stationary phase stationary early Culture the the
issue in the bioreactor the in issue s uel uniiain for quantification lupeol s
(30 days for AtLUS6 and 20 20 and AtLUS6 for days (30 , we compared the biomass biomass the compared we , : s
line LjLUS line other two lines showed showed lines two other were monitored over a a over monitored were AtLUS , well
which offers control control offers which - - 6 efrig lines performing , - 25 LjLUS ) Transgenic . , line did not not did line
with
- and 25 and and 25 system system a cell a the the the . This article isThis article by protected copyright. All rights reserved. complexes dynamic in assemble Accepted suited well allow metabolon create to approach scaffold synthetic investigation Further organisms enzyme heterologous enzymes the by overcome patterns expression Gene pathway biosynthesis micr enzymes heterologous multiple Articlefeasibility through Previous expressed pathway terpenoid complex enzymes: bound pathway and tricornutum have we Herein, CONCLUSIONS fitness. oalga. improv ,
ly
that si hs en hw t shown been has it as h itouto of introduction the
,
n rltv es i trs f cenn ad eei approach, genetic and screening of terms in ease relative and (Chappell et (Chappell al. 1995; etal. Moses 2014)
The results from this study p study this from results The
mono , o te atr prah a te plant the as approach, latter the for We show for the first time the possibility of introducing in introducing of possibility the timefirst the for show We
ed for the for
requires the expression of expression the requires
triterpenoid production in production triterpenoid
a lupeol synthase, a P450 enzyme and a NADPH reductase. This is This reductase. NADPH a and enzyme P450 a synthase, lupeol a -
and vrxrsin of overexpression demonstrate
produc wih a be can which ,
to that bypass native regulation that bypass native se
test the feasibility of having multiple enzyme fusions or using a a using or fusions enzyme multiple having of feasibility the test s suggest utreod production quiterpenoid t i o n
, single of d o result in higher sterol and triterpenoid levels in several in levels triterpenoid and sterol higher in result o to (Bassard et al. 2017) al. et (Bassard
that high h faiiiy f introducing of feasibility the reconstitute a plant secondary metabolite secondary plant a reconstitute
mlyd in employed
key - enzyme there is limited precursor availability, which could be be could which availability, precursor limited is there value sapogenins, without sapogenins, value rovide novel insights on insights novel rovide genes corresponding to corresponding genes
P. tricornutum P. MVA pathway pathway MVA o date to s We .
- CYP’s li .
ke complex ke . A prime candidate . Aprimecandidate HMGR would be the future future a be ahee i tee organisms these in achieved been has
.
have n htsnhtc microorganisms. photosynthetic in Moreover, we showed the feasibility feasibility the showed we Moreover,
localize . The . enzymes or the introduction of of introduction the or enzymes terpenoid terpenoid ,
therefore, betulinic acid (BA) acid betulinic es
the regulation of the sterol the of regulation the to three different membrane different three
evident (Dueber et al. 2009) al. et (Dueber
plant the membrane and can can and membrane the aha engineering. pathway P. tricornutumP.
demonstrated the the demonstrated transgenes impact on impact
of introducing introducing of
pathway in a in pathway
pathway
the most the
growth growth a plant a in , will will , P. is - This article isThis article by protected copyright. All rights reserved. ( phase log mid reaching until preculture as 25ml in inoculated then and days 5 for medium F/2 of 5mL tricornutum P. Strain andconditions growth MATERIALS AND METHODS producin productivity overall value contin screening enzyme include that microalga T independent lipid a in induced chemically be can biosynthesis unknown largely is diatoms of metabolism molecules. small of production the for feasible platform this before cultivation and control expression gene microo investigation and perspective engineering improving microalga this growing of Accepted study his Article - added molecules such as fucoxant as such molecules added u rganism ous batch system batch ous g platform. e
, therefore, ,
~ h product the as a chassis for the photo the for chassis a as 3 days) and then transferred then and days) 3 twrs nutil itcnlg exploitation biotechnology industrial towards s in
. CCAP1055/1
. tricornutum P. Substantial advances will need to be made for algae algae for made be to need will advances Substantial r isrcie ih ead o h etbihet f nw realm new a of establishment the to regard with instructive are
and economics and
represents pion represents
yield yield analyses
was grown in F/2 medium. Cells were Cells medium. F/2 in grown was at
, through
ag scale. large n engineering and hs first this ,
as well as well as towards the establishment of a renewable triterpenoid renewable a of establishment the towards
eering work validating the potential of this of potential the validating work eering production of value of production
h ipoes piiain and optimization bioprocess to - in and in eeain tan and strain generation
fresh medium with a 1:5 dilution. Cells were all were Cells dilution. 1:5 a with medium fresh lhuh hr i still is there Although use of a of use ; To date the regulation of the lipid and sterol sterol and lipid the of regulation the date To long chain chain long a recent study has shown that sterol sterol that shown has study recent a ,
oiiain n h cro sink carbon the on modification biorefinery approach for extraction of extraction for approach biorefinery
- added molecules. added PUFAs, manner manner
could
u rsls open results our o this for transfer could eoe commercially become substantial (Prioretti et al. 2017) al. et (Prioretti ih ead to regard with metabolic
likely red
photosynthetic photosynthetic F
uture from plate to plate from
improve the the improve
eukaryotic om for room path
studies , a
semi both both new way way
of of - - . ,
This article isThis article by protected copyright. All rights reserved. connected m 30 to (up pump centrifugal · illuminated was formation) manifold tube 6 a in consisting UK) Manchester, growth scale large For scale Large andshaking with temperature photobioreactor cell were brassica and lupeol monitor To every taken 10using the in minutes by 8 at kept was pH profile system analysis performance strain For Algem light 20 at grown s - Accepted1 Article ,
culture by eight 600W high pressure sodium lamps. sodium pressure high 600W eight by ( sub 60
s g with temperature with
rowth µmol (Algenuity - cultured of to a holding tank holding a to cultivation x10 2 of density °
C an
m analysis in New Brunswick New in
array of horizontal polycarbonate 50 mm 50 polycarbonate horizontal of array s − 2 . Light was set to 400 μmol photons m photons μmol 400 to set was Light . ih a with s
twice until they reached mid reached they until twice , UK , − 1 ). controlled bubbling with bubbling controlled
). a ,
Light was set to set was Light okn vlm o approximately of volume working and shaking and 6 fence
(200 L) (200 cells·mL pH control above pH control measurements asdescribed and OD trl productivity sterol 3 TM - ,
tl photobioreactor style
el wr gon n Algem® in grown were cells · -
. h built sensor.
incubator incubator The holding tank was continually sparged with sparged continually was tank holding The - - 1
1
kept constant at 21 °C 21 at constant kept ws loe b stainless by allowed was ) in fresh in F/2 medium and medium F/2 in fresh in 150
agitated at 120 rpm under white under rpm 120 at agitated
μmol photons μmol - 3 exponential phase, and then inoculated at inoculated then and phase, exponential
at a light intensity of 450 μmol photons μmol 450 of intensity light a at %
Continuous circulation of the culture via via culture the of circulation Continuous
CO n fr ee xrsin analysis expression gene for and 2
- in air. in
2
( · oyus nris n Services and Energies Bouygues s diameter tubes (36 tubes in total, in tubes (36 tubes diameter
- 1 5
and 120 and using the using
m 50 OD - 2
s L 740 -
labscale grown in Algem in grown 1 a used was
using the using nm te (1S pipework (316S) steel
rpm
measure ‘ sunlight
respectively. photobioreactor . ‘ A photostage photostage A sunlight ’ ments
fluorescent LED profile LED ®
a flux a labscale labscale .
,
’ were were
cells cells
LED LED The The m
of of - a 2 ,
This article isThis article by protected copyright. All rights reserved. in added was codon stop the without MtCYP716A12 for coding cDNA The . (72 sequence remaining the a and sequence membrane for encoding sequence the firstly MtCPR, with MtCYP716A12 of fusion the For 3 table ( polymerase 2015) al et & plasmids recipient final in were XM_003602850.2) sequence reference (NCBI AB181245), Goossens Alain accession number: pPhaT Plasmid design and transformation artificial sea ( water co temperature delivery air
Accepted Articlemposition, ~0 L (~10 synthe - . 1
Clue oioig wih nldd xgn auain p, odciiy and conductivity pH, saturation, oxygen included which monitoring, Culture .
( G - ·
spanning MtCYP716A12 enBank accession number: number: accession enBank s min
N ized F/2 nutrients (Varicon nutrients F/2 ew England ew measurement
(PSB (S - sequence cod sequence 1
) upplement as gBLOCKs as
NP178018.1 for I nstant -
VIB, Belgium) VIB, domain of MtCPR (1 MtCPR of domain
xgn eoa, while removal, oxygen ,
Biolabs according to the to according ( O G - al cean 692AA, enBank accession number DQ335781), number accession enBank
(IDT
a performed was i ng for a for ng ) Fig and ble and , USA , , Leuven, Belgium) Leuven, , . .
Hitchin, UK Hitchin,
Sequence
1). A 214 qua ).
Where necessary, PCRs were performed with Q5 Q5 with performed were PCRs necessary, Where r flexible linker GSTSSGSG (λ) was added in added was (λ) GSTSSGSG linker flexible
-
marker gene sequence was a gift from the group wasagift of fromthe genesequence marker 20179bp , United Kingdom United , - 71 AA, 71 AF219942) design rules of the the of rules design s f s , ).
pH a
nat o All primers used are listed in listed are used primers All i a rflx interface. 3 Profilux a via r
) creating a cDNA encoding cDNA a creating ) LjLUS 1 r : (GenBank accession number: number: accession (GenBank : - -
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This article isThis article by protected copyright. All rights reserved. obtained were fromStandards Extrasynthese using performed saponification quantification For described was cyclodextrin methyl beta containing 5mM cyclodextrin 4,200 centrifugation until grown were lines cell All GC fresh 20 at incubated 24h in placed were plates n NaCl) of 1.7% described of transformation Biolistic version of λΔ7 sequence the to assembly o urseothricin
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(Sigma Aldrich, DE) was added to 25 mL cell culture to a final concentration of of concentration final a to culture cell mL 25 to added was DE) Aldrich, (Sigma prior
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This article isThis article by protected copyright. All rights reserved. of correlationwas 1.2 agricolae the using 3.43 R in computed were tests Tukey and 2001) Schmittgen Manager CFX in implemented as method ΔΔCt the using performed was Analysis Quant 2017) al. earlier described as genes reference as used were genes UBQ and RP3a the and UK manufacture the protocol. following (Thermo) TriReagent using extracted were DNA and RNA numberCopy analysisand ex mRNA 290°C Oven 320°C. to set was line Aldrich (Sigma column GC All and aboveharvested processedasdescribed for the of mL 400 above, For
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it (50°C/minute ramp), after which to (50°C/minute ramp),after itwasramped 300 - S ape wr rn n Tem TRACE Thermo a on run were samples MS ative PCR was perfo was PCR ative Te sa The . First strand cDNA synthesis was performed using MM using performed was synthesis cDNA strand First
detection, afterquickscreenbeta methylcyclodextrinmethoddetection, with
(Biorad determined by calculating at determined by e rmr wr ue a normaliz as used were primers me , UK , oiie ie wr gon ni sainr pae (~ phase stationary until grown were lines positive , UK , ). The in The ). rmed with JumpStart SYBR green ReadyMix (Sigma Aldrich (Sigma ReadyMix green SYBR JumpStart with rmed
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All primers used are used primers All jector temperature was set to set was temperature jector pression analysispression set to 120°C and subsequently increased to to increased subsequently and 120°C to set - statistic l upeol quantification analysis. upeol quantification
- listed in supplementary table 3 table supplementary in listed C SI qarpl wt a SLB a with quadrupole DSQII GC
using the formula: r fr h gN CV analysis. CNV gDNA the for ers analysis, analysis, ° C at (1°C/ minute). minute). C at(1°C/ - u 8 package 8 280°C LV (N LV cDNA was diluted 1/20 diluted was cDNA
ew England Biolabs, Biolabs, England ew and the MS the and . The significance The .
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transfer ANOVA ANOVA
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5MS 5MS
r ). ). s This article isThis article by protected copyright. All rights reserved. Accepted P.Arendt, etal., biology 2016. andnew of Synthetic forproduction natural Apt, K.E., Kroth A. Anterola, et al.,2009.Productionof taxa C.M.Andre, etal.,2016. cyclasesMultifunctional oxidosqualene P450 andcytochrome REFERENCES used forpartsof thisstudy. Article a Algenuity, by Spicer employed of were division MM and APS GPL, GS, JS, SD, study the of time the At Conflicts Program Framework FP7/2007 Seventh Union's European the of product final to discovery from Products Value High Plant [KBBE.2013.3.1.01] Theme the from funded partly been has Moses support technical for Pallister Denise and Sizer Sam thank We ACKNOWLEDGMENTS terpenoids inphotosynthetic organisms.terpenoids pp.572 diatom Phaeodactylum trico patens. pp.1279 involved biosynthesisof inthe fruittriterpenicacids. apple and Jacob Pollier Jacob and
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d for the prevention and treatment of cancer treatmentd forofcancer and HIV the prevention and Posewitz, M.C., 2015. Toward a photosynthetic microbialphotosynthetic Posewitz, M.C.,2015.Towarda , 27(8), - type precursors type in Saccharomyces cerevisiae. , 2, p.21. pp.5 , 114(11), pp.2528 Photosynthesis research Photosynthesis
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This article isThis article by protected copyright. All rights reserved. Accepted M.L.Hamilton, 2016.et al., Hamberger, B. &2013. Bak,S., PlantP450s drivers asversatile for of evolution species Gimpel, J.A.etal.,2015.In MetabolicEngineering of Eukaryotic Fukushima, E.O. etal.,2011. M. Fuhrmann, et al.,2004. in dynamic genes expression Monitoring ofnuclear H.etal., Franziska, 2011. Microalgae foras bioreactors production. bioplastic ArticleFalciatore, aetal., 1999. of Transformation GenesinMarine NonselectableReporter M.Fabris, thesterolbiosynthesis etal.,2014.pathwayofthediatom Tracking F.,Restovic,Erpel, & F. polyunsaturated fatty acidspolyunsaturated by trophically marine converted diatom phaeodactylum &rendertype=abstract. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3538417&tool=pmcentrez B,BiologicalSeries sciences chemicalspecific diversity. Challenges withGreat Diversity.Come pp.1 ,6(December), triterpenoid biosynthesis. luciferaseChlamydomonas byusingasynthetic reinhardtii rep http://w Diatoms. http://www.ncbi.nlm.nih.gov/pubmed/24996048. Phaeodactylum tricornutum 16(1), p.29. chlamydomonas animalnutrition. formonogastric reinhardtii pp.123 – 129. ww.ncbi.nlm.nih.gov/pubmed/10383998. Marine biotechnology(NewMarine York,N.Y.)
Arce Heterotrophic production of omega production Heterotrophic
- Johnson, P.,Development of 2016. phytase CYP716A subfamily CYP716A members are multifunctional oxidases in Plant and Cell PhysiologyCell Plant and Philosophical transactions oftheSocietyPhilosophical transactions Royal o , 368(1612), p.20120426. at: Available . The Newphytologist , 1(3), pp.239 . Available at:
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This article isThis article by protected copyright. All rights reserved. Kim, D.H. ofactivityet al.,2005. synthase isoflavone byco Enhancement AcceptedKhakimov, B. etal.,2015. Identification organization andgenome ofpathwaygenes saponin K. Jensen, & Møller, B.L., 2010.NADPH Plant Jennewein, S. etal., of2005. Coexpressioninyeast P450 Taxuscytochrome with reductase Husselstein Article Z. Huang, et al.,2015.dammarane Production of Hsieh, M. Herrera, J.B. etal., characterization1998. CloningArabidopsis of thalianalupeol and the Hempel, F.etal.,2011. asproteinfactories: Algae expressionantibody andthe of a human benthamiana. from awildcrucifer, and theirusefo Phytochemistry bioengineering cytochrome P450 involved oxygenases inTaxolbiosynthesis. biologymolecular ofin yeast 2,3 pp.106 dammarenediol p.641 LP Plastid Nonmevalonate Pathway ofIsoprenoidBiosynthesis. synthase gene. p.e28424. respective antigen inthediatomPhaeodactylumtr tricornutum. - H. & Goodman, H.M., 2005. The Arabidopsis IspH Homolog H.M.,H. &TheArabidopsisIspHIsInvolv 2005. Goodman, – – Muller, T.,Muller, H. Schaller, & P., cloning Benveniste, 2001.Molecular andexpression 114. - 653. http://www.plantphysiol.org/content/138/2/641.abstract. Available at:
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r transient productionr transient ofsaponins inNicotiana
- cytochrome P450cytochrome oxidoreductases. - type sapogeninstype inrice by exp – – 490. 1911. icornutum.
Plant Physiology abidopsis thaliana. abidopsis thaliana. Biotechnology and Biotechnology PLoS One Plant Science - expression of expression P450 , 6(12), ed in the ed inthe ressing the , 239, , 138(2), Plant
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gura, M.J., M.M. Meyer, & S.P., Matsuda, 2000.thalianaLUP1 Arabidopsis converts Accepted Article Biocatalysis. Plant Science http://www.sciencedirect.com/science/article/pii/S0168945205003018. cyclases of Lotus japonicus. specificity in planttriterpenesynthases. ,pp.1 pp.405 132, pp.60 5alpha 5(1), pp.1 Regulation of theSupplyCommonPrecursors in Plant Cells. for thegreenChlamydomonasmicroalga reinhardtii. , Algae. 9(4). microbiology andbiotechnology - Concep
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PLoS genetics Frontiers in , 24(3), Journal of Journal , , This article isThis article by protected copyright. All rights reserved. for thecommonprecursor 2,3 Accepted enzymes pathway sapogenin plant in The introduced reductase. NADPH coenzyme its with along enzyme, family P450 cytochrome a through acid betulinic to modified further be can lupeol sapogenin 2,3 plant the pathway the In brassicasterol. to leading and (IPP) pyrophosphate precursor isopentenyl the from starting diatoms in metabolism sterol of representation Schematic Figure 1. Engineering LEGENDSFIGURE Zhou, C.etal., ofbetulinicacid2016. biosynthesis in Improvement employingyeast multiple Article R.C.,Zangar, Davydov, & D.R. thatregulate Verma,S.,2004.of production Mechanisms Z.etal., Xue, 2012. cyclases evolution Divergent inplants.of oxidosqualene Wor Work, V.H. al.,2013. et Biocommoditiesfromphotosynthetic microorganisms. k, V.H. et al., 2012. Improving photosynthesis and metabolic networks k, V.H.al.,2012. andmetabolicnetworks forthe et Improving photosynthesis strategies. 199(3), pp.316 reactive oxygen species by cytochrome P4 Phytologist http://www.ncbi.nlm.nih.gov/pubmed/22172528 [Accessed July biotechnology competitive of phototroph production Environmental Progress Energy and Sustainable P. tricornutum P. BMC Biotechnology , 193(4), pp.1022 -
xdsuln peusr is precursor oxidosqualene , 23(3), pp.290 – 331. P. tricornutum
will, therefor will, - oxidosqualene. – – 1038. 7. Available at: 7. Available , pp.1 by introducing
– 9. e, compete with the native cycloartenol synthase cycloartenol native the with compete e, - derived biofuels.derived
ylzd o uel b a uel ytae the synthase; lupeol a by lupeol, to cyclized 50. Toxicology and pharmacology applied Toxicology , 32(4), pp.989 a plant
sapogenin pathway. Current opinion in Current – 1001.
11, 2014].
New
, This article isThis article by protected copyright. All rights reserved. Acceptedexpression of brassicasterol was oftheWTor detected inthe medium either LjLUS LjLUS from cellpellet andtheLjLUS ofWT (diamonds) only detectedLupeol was intheLjLUS ofbrassicasterolproductivity percell for (diamonds) thewildtype andLjLUS inlabmedium Figure 4. productivity Lupeol in batch 400mL cultures. where means are notstatistically different according to a the LjLUS geometric average ofthe lines showing expression of the ArticleFigure 3. withtheirbest peaks these for spectra mass molecules, terpenoid unidentified * molecule; diterpene unidentified by dihydroxylupane black. in standard AtLUS lines transformant in production Lupeol 2. Figure standard deviation from
- expression cassettes 25 is shown by the triangles in the cell (full and 25 bythetrianglesincell line) shown is Expression and copy number oflupeol producing strains. (A)
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diinl peaks: Additional Triplicate cultures grownTriplicate were inF/2 Identical - 25 transformant line (WT astransformant line (WT 25
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the (dashed medium line). No
( Copy of number integrated LjLUS
letters denote groups (grey) . Brassicasterol extracted . Brassicasterolextracted . Cell density and . Celldensity
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(squares). (squares). . Gene . Gene α e). = 0.01.
3 of six Lupeol , 20 β,
- This article isThis article by protected copyright. All rights reserved. D C this paper Phaeodactylum tricornutum Taroda Hiroyuki and B p. 419 Phaeodactylumtricornutum A References for panelA: create productsinmicroalgae) costoffsets.carotenoids (unique tric for biomass of Cost efficiency. extraction of levels equal assuming be to estimated are co relevant commercially from triterpenoids for procedure extraction from triterpenes of approach biorefinery Fig where means are notstatistically different according to a represent correspond and negative control) monitored duringculturingtimeandrelative to the referencegenes
Yu S Accepted Yongmanitchai *W. Article ure o .
UBQ - rnutum . Tibbetts M. Hong Yang, Lei Du, Masashi Hosokawa, Kazuo Miyashita, Yume Kokubun, Hisayoshi Arai Masashi Hosokawa, Hisayoshi Arai Yang,LeiDu,Kazuo Miyashita,YumeKokubun, Hong - 425 0099 5 . . Red arrows indicate theday maximum. Redarrowsindicate lupeol when isobserved productivity the ceai rpeetto of representation Schematic ing
is higher (produced in Europe, Nec Europe, in (produced higher is
standard deviationfrom3 biologicalreplicates.
to mid - , 2240/91/020419 J .
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- Fatty Acid and Lipid Class Composition of the Microalga Microalga the of Composition Class Lipid and Acid Fatty products compared to the yeast yeast the to compared products
. . ad rwh f n Omega and of Growth Ward P. 0. ential growthential phase J. Oleo66, Sci. (4) 363 under Different Culture Conditions Culture Different under ,
P. - Santosh 07$02.00/0
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n J. and P. tricornutum tricornutum P. . P.
ton), but market sizes of lipids omega 3 and and 3 omega lipids of sizes market but ton), as describ tric Lall - 368 (2017) o
hmcl opsto ad nutritional and composition Chemical rnutum
post hoc ed and S. cerevisiae S.
Identical letters denote groups in the text. Allbarsin thetext. error
iutnosy slts more isolates simultaneously DOI
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Appl Fty cd rdcin by Production Acid Fatty 3 S. cerevisiae cerevisiae S.
Tukey test with : 10.5650/jos.ess16205 . E nv. . Cost of extraction extraction of Cost .
Micro. biomass. The The biomass. , Feb. 1991, 1991, Feb. , α = 0.01. RP3a
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This article isThis article by protected copyright. All rights reserved. ISP cytidylyltransferase, phosphate Enzyme m in involved reductase Hydroxymethylglutaryl grey. in indicated are LjLUS the LjLUS and WT for map P. type wild the Figure Biotech p Förster, G Comparison ofmethods different F Mar E References for panelB: Phycol (2015) 27:1109 prop
outo o btlnc cd n is lupane its and acid betulinic of roduction International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol (IJEAB) Biotechnology and Agriculture Environment, of Journal International onooxygenase . Lamac M. E. Czarnotta, M. Dianat, M. Korf, F. Granica, J. Merz, J. Maury, S. A. Baallal Jacobsen, J. J. Jacobsen, Baallal A. S. Maury, J. Merz, J. Granica, F. Korf, M. Dianat, M. Czarnotta, E. Acceptedtricornutum Article RP3a - erties of freshwater and marine microalgal biomass cultured in photobioreactors in cultured biomass microalgal marine and freshwater of erties Apr 6 .
ivle i the in involved s .
-
Bioeng . E. B.
mRNA Expression of sterol biosynthesis enzymes during a five day time course in course time day five a during enzymes biosynthesis sterol of Expression mRNA and and 2017 http://dx.doi.org/10.22161/ijeab/2.2.22456 ISSN: ;
IDI
rsiatrl biosynthesis: brassicasterol K UBQ (WT) , indicating the gene selected for mRNA expression analysis and relative heat heat relative and analysis expression mRNA for selected gene the indicating , ad . S J. and a Ebert, - . 114, 2528 Q: spney dpopae isomerase diphosphate isopentenyl SQS: seo dhdoeae C4 trl euts, 2 seo desaturase. sterol C22 reductase, sterol C24 dehydrogenase, sterol ,
eeec genes reference and the LjLUS the and
– n L. and - -
1119 25 line. 25 o synthase; CoA
- A 2538 (2017)
bdr rotrl eemnto in determination Ergosterol jbidor
plastidial
. Bl M. Enzymes involved in the cytosolic MVA pathway: HMGS: HMGS: pathway: MVA cytosolic the in involved Enzymes Values are the expression the are Values . -
. Biotech 25 line 25 - ank. E: 4 E: rncit nt infcnl dfeet ewe w and wt between different significantly not Transcripts
M - DOI 10.1002/bit.26377 diphosphocytidyl MR 3 HMGR: . Fermentation and purification strategies for the the for strategies purification and Fermentation EP .
Schematic representation of sterol metabolism in in metabolism sterol of representation Schematic Tech.
-
PtOSC ye rcros in precursors type pathway: 11, 723 - : hydroxy o ioqaee cyclase, xidosqualene
– SD: 2 ISPD2: 725 - 2 relative to the geometric avera geometric the to relative - - C - 3 s - ( - uln synthase qualene methyl 1997
methyl acaoye cerevis Saccharomyces acaoye cerevisiae Saccharomyces - - 1878Page |558 C )
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methyl - - D glutaryl - erythritol kinase erythritol - D - - coenzyme coenzyme rtrtl 4 erythritol methyls . -
2, Issue 2, Enzymes Enzymes
J Appl J terol terol ge of of ge iae .
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This article isThis article by protected copyright. All rights reserved. from synthase ( ( a/c chlorophyll fucoxanthin native of control under were gene expressed for used plasmids of Illustration 1. Figure Supplemental SUPPLE aldehyde, and acid. betulinic conve is which intermediate, betulin to leads C28 at oxidation The reaction. Betulin. 3. Lupeol; 2. Brassicasterol; 1. indicated: as LjLUS in MtCYP716A12λΔ72CPR protein fused harboring construct with line transformant (purple); background WT t harboring constructs with line transformant of Figure DMAP: C MEP pyrophosphate, the in mentioned are numbers e non the box, yellow the AtLUS FCPA/LHCF1 runcatula runcatula gnee sri LjLUS25. strain ngineered - Accepted Articlemethylerythritol WT (blue), (blue), WT ) and ) 7 Dimethyl MENTAL LEGENDS . Betulin production in production Betulin . yohoe P450 cytochrome
zeocin resistance ( resistance zeocin ) promoter. ) L. japonicus L. L allyl pyrophosphate, j LUS
CDP , : - 25 2 - C
- - otiig ny the only containing A. -
methyl aie uel ytae from synthase lupeol native MEP:
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Expression vector containing lupeol synthase from synthase lupeol containing vector Expression
Dashed lines indicate multiple reactions. Gene accession accession Gene reactions. multiple indicate lines Dashed (Mt ble P. tricornutum P. - ) and ) 4 CYP716A12) D results section. Precursor Precursor section. results r - ) - diphosphocytidyl
erythritol 4 erythritol encoding genes encoding
G3P: Glycerol 3
zeocin resistance ( resistance zeocin - 25 background (light blue); standards (black). Peak (black). standards blue); (light background 25
and - . japonicus L. transformant lines. Panel A. Panel lines. transformant phosphate . japonicus L. yohoe 40 euts (Mt reductase P450 cytochrome - - B. Phosphate, Pyr: Pyr: Phosphate, Pyruvate 2 - ae B. Panel
C Expression vector containing lupeol lupeol containing vector Expression
Lotus - ble P. tricornutum P. methyl , , CDP , , r ) l po synthase upeol
encoding genes encoding abbreviations l japonicu po synthase upeol
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binding protein A protein binding : P450 GC rted to rted . IPP: Isopentyl Isopentyl IPP: . (LjLUS) C.
- - MS analysis analysis MS (LjLUS) phosphate, phosphate, A. thaliana A. Expression Expression - reductas
CPR) betulin ( , red
- M. M. All 2 in ); e -
This article isThis article by protected copyright. All rights reserved. Genetics Evolutionary Molecular MEGA7: (2016). K. Tamura and G., Stecher S., Kumar 2. AcceptedEvolutionBiology and18:691 maximum a using families protein evolution multiple from protein derived of model empirical general A (2001). N. Goldman, and S. Whelan, 1. LjLUS: roseus Catharantus annua Artemisia AaOSC2: [2]. MEGA7 in conducted were analyses Evolutionary dataset. final the in positions Ther position. any at allowed were bases ambiguous and data, missing gaps, alignment 5% than fewer is, That eliminated. were coverage site 95% than less with positions All site. per substitutions of number the in measured lengths branch with scale, to dr is tree The value. likelihood log superior with topology the selecting then and model, Neighbour Initia branches. Article to the is shownnext clusteredtaxatogether theassociated inwhich of trees percentage The highest log likelihoodby Likelihood Maximum method ofWhelanandmodel Goldman [1]. Figure2 Supplemental nourseothricin ( to fused of gene encoding engineered containing vector Expression gene. containing vector Expression containing vector Lotus japonicus Lotus E.
xrsin etr otiig oretrcn ( nourseothricin containing vector Expression - . truncatula M. Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT JTT a using estimated distances pairwise of matrix a to algorithms BioNJ and Join te() o te ersi sac wr otie atmtcly y applying by automatically obtained were search heuristic the for tree(s) l nat AtLUS: , r . truncatula M. ) resistance ERG7: ,
OeLUS: .
Molecular Phylogenetic tree of selected plant OSCs with the the with OSCs plant selected of tree Phylogenetic Molecular
yohoe 40 euts ( reductase P450 cytochrome rbdpi Thaliana Arabidopsis - . cerevisiae S. 699. M. truncatula M. Olea Olea europea
encoding gene.
yohoe 40 ( P450 cytochrome
aotrl yls, G cyclase, Lanosterol , ToLUS:
cytochrome P450 reductase ( reductase P450 cytochrome
BpLUS: , Taraxacum officinale MtCYP716A12
nat - MtCYP716A12λΔ71MtCPR M. truncatula M. r likelihood uluu lanceolate Bupleurum resistance ) gBAS: ) encoding gene. gene. encoding ) approach. Molecular Molecular approach. e were a total of 708 708 of total a were e lcrhz Glabra Glycyrrhiza
enc
cytochrome P450 cytochrome MtCPR oding gene. gene. oding ) encoding encoding ) , CrBAS: CrBAS: , and ) awn awn D. D. F. ,
This article isThis article by protected copyright. All rights reserved. biological three replicates.standard deviationfrom LjLUS (WT). strain type wild UTEX646 A. photobioreactors Figu Supplemental AtLUS AtLUS in 1 after * peak Fourth molecule). like sterol (unidentified AtLUS and W from H. from peak hydroxylupane LjLUS from peak 3 Figure Supplemental Evolution 39:783 Con (1985). J. Felsenstein 3. versionAnalysis Biologybigger datasets.Molecular andEvolution33:1870 7.0 for
Accepted Article Growth performance in Algem® photobioreactors of the AtLUS the of photobioreactors Algem® in performance Growth Betulin peak of peak Betulin - - 5 tan oprd o h CA15/ wl tp sri (WT). strain type wild CCAP1055/1 the to compared strain 25 6 sterol(unidentified , LjLUS T, LjLUS+MtCYP716A12+MtCPR. - (ndniid diterpene (unidentified 6 - rm AtLUS from P. 5 n AtLUS and 25 - - 791 . 25.
hr pa * fe 1 n AtLUS in 1 after * peak Third - LjLUS_MtCYP716A12 e 4 re like molecule). like
B. .
.
GC
uel ek f standard. of peak Lupeol Growth performance of the LUS transgenic lines in Algem® Algem® in lines transgenic LUS the of performance Growth - - - fidence limits on phylogenies: An approach using the bootstrap. bootstrap. the using approach An phylogenies: on limits fidence MS ionization pattern for lupeol and betulin peaks betulin and lupeol for pattern ionization MS like molecule). 6. - - 6 (unidentified sterol (unidentified 6 , dniid s campesterol. as identified 6, E. B.
uel ek of peak Lupeol Growth performance in Algem® photobioreactors of the the of photobioreactors Algem® in performance Growth O.
- Second peak * after 1 in AtLUS in 1 after * peak Second ie molecule). like λΔ G. 71MtCPR.
Lupeol peak of peak Lupeol -
(ndniid sterol (unidentified 6 LjLUS+MtCYP716A12+MtCPR. LjLUS+MtCYP716A12+MtCPR.
- like molecule). like I. C. N.
Betulin peak of standard. of peak Betulin
uel ek f AtLUS of peak Lupeol
is pa * fe 1 n AtLUS in 1 after * peak First LjLUS_MtCYP716A12 M. Peak - R. 6 line compared to the to compared line 6 -
6 (unidentified st (unidentified 6 Fifth peak * after 1 in in 1 after * peak Fifth b ro br represent bars Error rm T LjLUS WT, from - like molecule). molecule). like λΔ - - 1874. 6. . F. 71MtCPR. 71MtCPR. A. L.
D.
Betulin Peak Lupeol Lupeol
erol 2 - - 3 25 Q.
- 6 a -
This article isThis article by protected copyright. All rights reserved. quantification harvested weight dry cell total and harvest at densities cell harvested, 550L T Supplementary three biological replicates. ofliter culture from previous semi transformant lines. Supplementary T 3the PBRforasecondharvest(455L)after days. v same the and days, 12 after harvested first were 373L LjLUS_MtCYP716A12_MtCPR_6, For medium. fresh with PBR the restarting after dying started culture the then 29, day at harvested been have 285L LjLUS_25 For days. 21 int medium F/2 fresh with replenishedvolume same the culture. For dots restart green the AtLUS point, time harvest indicate dots Red photobioreactor. tubular 550L in grown lines three Figure Supplemental transformation. background. A. photobioreactors 5 Figure Supplemental
Accepted Article rwh efrac o te eetd MtCYP selected the of performance Growth
tubular photobioreactor (PBR) photobioreactor tubular , andper gram ofdryweight per gram dry weight wasalso gramdry per determined B. Error bars represent bars standarddeviationf Error be . Scale 2. able
able 1. Quantification oflupeol accumulation in thebestperforming .
rwh efrac o te MtCYP716A12 the of performance Growth -
quantitative analyses. Quantitiesquantitative analyses. werenormalized cellnumber per Lupeol extracted from cell pellet of extracted frompellet bestperformingst Lupeol cell 6 . .
Growth performance in 550L tubular PBR. tubular 550L in performance Growth rwh efrac o te eui tra Betulin the of performance Growth
- u cniin ad specification and conditions p
used for pilot scale production scale pilot for used - 6 approximately 454L was harvested at day 17, and 17, 454L6 harvestedatday approximately and was . Error bars represent standard deviation from olume replenished with fresh F/2 medium into medium F/2 fresh with replenished olume -
MtCPR line transformed into the LjLUS the into transformed line MtCPR . o the PBR for a second harvest after after harvest second a for PBR the o
rom three biological three replicates.rom .
G rowth rowth nsgenic lines in Algem® Algem® in lines nsgenic . Strain, liter of biomass of liter Strain, . - λ Growth curves for the for curves Growth - Δ71
are shown. are p rains, selected arameters P LLS co LjLUS CPR Lupeol Lupeol f the of ,
per per
- 25 -
This article isThis article by protected copyright. All rights reserved. Accepted Article Sup Supplementary as PCRprimersfor of domestication golden partsandQPCR. gate T Supplementary plementary Table5 T able 4 able 3. Oligonucleotides used in this study. this in used Oligonucleotides 3. able . .
Repeated measuresRepeated sign Sources for analysis Sources theeconomic
ificance ofificance shown thegenes in F
.
List of oligo of List
nucleotides used used nucleotides igure 6 igure .
This article isThis article by protected copyright. All rights reserved. Accepted Article
This article isThis article by protected copyright. All rights reserved. Accepted Article
This article isThis article by protected copyright. All rights reserved. Accepted Article
This article isThis article by protected copyright. All rights reserved. Accepted Article
This article isThis article by protected copyright. All rights reserved. Accepted Article
This article isThis article by protected copyright. All rights reserved. Accepted Article
This article isThis article by protected copyright. All rights reserved. Accepted Article