Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene

Gao Xue, Yuan Xiao-Xi, Shi Zhen-Yu, Guo Ying-Ying, Shen Xiao-Wen, Chen Jin-Chun, Wu Qiong, Chen, Chen Guo-Qiang

Description

Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2 Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2 Ps is limited with very low PHA yield. Results To improve the production of PHA with a wide range of monomer compositions in E. coli , a series of optimization strategies were applied on the PHA synthase PhaC2 Ps . Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli . The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2 Ps , the optimized PhaC2 Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2 Ps O with a hairpin structure in the 5’ untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2 Ps . Conclusions The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2 Ps . These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.

Sujets

• PHB
• Polyhydroxyalkanoates
• Hairpin
• Escherichia coli

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 13
• Langue: English

Extrait

Gao
etal.MicrobialCellFactories
2012,
11
:130
http://www.microbialcellfactories.com/content/11/1/130

RESEARCH

OpenAccess

Productionofcopolyestersof3-hydroxybutyrate
andmedium-chain-length3-hydroxyalkanoates
by
E.coli
containinganoptimizedPHA
synthasegene
XueGao
1
†
,Xiao-XiYuan
1
†
,Zhen-YuShi
2
,Ying-YingGuo
1
,Xiao-WenShen
1
,Jin-ChunChen
1
,QiongWu
1*
andGuo-QiangChen
1,3*

Abstract
Background:
Microbialpolyhydroxyalkanoates(PHA)arebiopolyestersconsistingofdiversemonomers.PHA
synthasePhaC2
Ps
clonedfrom
Pseudomonasstutzeri
1317isabletopolymerizeshort-chain-length(scl)
3-hydroxybutyrate(3HB)monomersandmedium-chain-length(mcl)3-hydroxyalkanoates(3HA)withcarbonchain
lengthsrangingfromC6toC12.However,thesclandmclPHAproductionin
Escherichiacoli
expressingPhaC2
Ps
is
limitedwithverylowPHAyield.
Results:
ToimprovetheproductionofPHAwithawiderangeofmonomercompositionsin
E.coli
,aseriesof
optimizationstrategieswereappliedonthePHAsynthasePhaC2
Ps
.CodonoptimizationofthegeneandmRNA
stabilizationwithahairpinstructurewereconductedandthefunctionoftheoptimizedPHAsynthasewastestedin
E.
coli
.Thetranscriptwasmorestableafterthehairpinstructurewasintroduced,andwesternblotanalysisshowedthat
bothcodonoptimizationandhairpinintroductionincreasedtheproteinexpressionlevel.Comparedwiththewildtype
PhaC2
Ps
,theoptimizedPhaC2
Ps
increasedpoly-3-hydroxybutyrate(PHB)productionbyapproximately16-foldto30%of
thecelldryweight.Whengrownondodecanoate,therecombinant
E.coli
harboringtheoptimizedgene
phaC2
Ps
O
withahairpinstructureinthe5
’
untranslatedregionwasabletosynthesize4-foldmorePHAconsistingof3HBand
medium-chain-length3HAcomparedtotherecombinantharboringthewildtype
phaC2
Ps
.
Conclusions:
ThelevelsofbothPHBandscl-mclPHAin
E.coli
weresignificantlyincreasedbyseriesofoptimization
strategiesappliedonPHAsynthasePhaC2
Ps
.Theseresultsindicatethatstrategiesincludingcodonoptimizationand
mRNAstabilizationareusefulforheterologousPHAsynthaseexpressionandthereforeenhancePHAproduction.
Keywords:
PHB,Polyhydroxyalkanoates,PHAsynthase,Codonoptimization,Hairpin,Escherichiacoli

Background
[7,8].Onthebasisofthecarbonchainlengthsofmono-
Polyhydroxyalkanoates(PHA)areafamilyofbiopolyestersmers,PHAareclassifiedasshort-chain-lengthPHA(scl
accumulatedbymanybacteria,andhavebeenstudiedtoPHA)andmedium-chain-lengthPHA(mclPHA)consist-
meetthevariousdemandsinchemical,materialandspecialingofthreetofiveandsixormorecarbonatomsintheir
industries[1-6].ThestructuresandpropertiesofPHAcanmonomers,respectively[7,9].ThedifferencesinPHA
beadjustedbycontrollingtheirmonomercompositionsmonomersandcompositionscanstronglyaffecttheprop-
ertiesandqualitiesofthepolyesters[10-12].Generally,
*Correspondence:[e-mail];[e-mail].
PHAcopolymersconsistingofsclandmclmonomersare
edu.cn
†
Equalcontributors
consideredtohavemorepotentialsforapplicationsdueto
1
MOEKeyLabofBioinformatics,DepartmentofBiologicalScienceand
theirsuitablepropertiesincludingtensilestrength,Young
’
s
Biotechnology,SchoolofLifeScience,Tsinghua-PekingCenterforLife
modulus,elongationatbreakandsoon[13].Particularly,
Sciences,TsinghuaUniversity,Beijing100084,China
3
CenterforNanoandMicroMechanics,TsinghuaUniversity,Beijing100084,China
thesclandmclPHAcopolymersconsistingofhighmolar
Fulllistofauthorinformationisavailableattheendofthearticle
percentageofsclmonomers(suchas3HB)andlowmolar
©2012Gaoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

Gao
etal.MicrobialCellFactories
2012,
11
:130
http://www.microbialcellfactories.com/content/11/1/130

percentageofmclmonomershavebetterapplicationprop-
ertiesincludinghighmeltingtemperatureandtoughnessas
wellasrapidcrystallizationprocess[14].Manybacteria
suchas
Aeromonashydrophila
and
Ralstoniaeutropha
werereportedtobeabletoproducesclandmclPHAwith
differentcompositionsandyields[15,16].However,there
arestilldifficultiestocontrolthePHAmonomercomposi-
tionswhenindustrialfermentationprocessesareconducted
forproductionofPHA[8].
OneofthekeyfactorsthatdeterminePHAmonomers
compositionisthesubstratespecificityofthePHAsyn-
thase(PhaC).TheproductionofsclandmclPHA
requiressynthaseswithrelativelywidesubstratespecifi-
cities,suchasPhaCfrom
Aeromonascaviae
[17,18]and
PhaC2
Ps
from
Pseudomonasstutzeri
1317(named
PhaC2
Ps
)[19].ThesePHAsynthaseshavebeenwell
characterizedandexpressedforproductionofscland
mclcopolymers[17,19].Forexample,whenexpressedin
aPHAsynthasegene
phbC
Re
negativemutant
R.eutro-
pha
PHB-4,PhaC2
Ps
couldconferonthehoststrainthe
abilitytosynthesizePHAwithmonomercompositions
adjustablebyalteringcarbonsources[19].Furthermore,
toachieveahighmolefactionof3-hydroxybutyrate
(3HB)monomerinscl-mclPHAcopolymersforbetter
thermalandmechanicalproperties,specificpointmuta-
genesiswassuccessfullyapplied[20],leadingtomutated
PHAsynthasePhaC2
Ps
QKSTthatincreasedPHAaccu-
mulationupto42wt%consistingof93mol%3HBin
R.
eutropha
PHB-4.However,allofthePhaC2
Ps
mutants
havenotyetbeencharacterizedin
E.coli
whichisa
well-developedcellfactoryformanyfineproducts[21].
E.coli
wassuccessfullyexploitedforsclPHA[22]and
scl-mclPHAproduction[23,24].Particularly,pathways
wereconstructedtoachievescl-mclPHAaccumulationin
E.coli
usingrelatedorunrelatedcarbonsources[25-27].
However,theoverallPHAyieldsweregenerallylowprob-
ablyduetoinadequatesupplyofPHAprecursors,and/or
lowersynthaseactivityduringthepolymerizationprocess.
ThestabilityofmRNAisoneofthemostsignificant
factorsthataffectlevelsofproteinsynthesis,inthiscase,
PHAsynthasesynthesis.Itwasreportedthatthesec-
ondarystructureswithin5
’
untranslatedregions(UTRs)
ofprokaryoticmRNAcouldactasmRNAstabilizers,
preventthemfromfastdegradationbyRNasesand
thereforepromotetranslation[28].Variousrationally
designedsynthetic5
’
hairpinstructureshavebeeninves-
tigatedandtheirhalflivesandeffectsinmRNAstability
weremeasured[28,29],anditissignificanttoadddiffer-
entmRNAsecondarystructuresinthe5
’
UTRregion
forcontrollingtheexpressionofrecombinantgenes.On
theotherhand,species-specificvariationsincodon
usagearegenerallyconsideredoneofthemajorfactors
thataffectheterologousproteinexpressions.Ifthecon-
centrationsofthe
E.coli
tRNAsfortherarecodonsare

Page2of10

insufficienttooptimallytranslatemRNA[30],codon
optimizationcouldbeeffectivetoenhanceproteinex-
pression[31,32].
Toachievehighscl-mclPHAproduction,thefunc-
tionsofthewildtype
phaC2
Ps
anditsrecombinantsin
E.coli
wereevaluatedinthisstudy,andcodon
optimizationandmRNAstabilizationstrategywere
employedtoenhancePHAsynthaseexpression.There-
combinant
E.coli
expressingtheoptimizedPhaC2
Ps
was
proventobeabletoproducescl-mclPHAmuchmore
effectivelythanthewildtypedid.
Results
AnalysisandoptimizationofthemutatedPHAsynthase
PhaC2
Ps
QKST
Inpreviousstudies,thescl-mclPHAproductionabilities
ofseveralmutantsofPhaC2
Ps
werestudied[20].Adouble
mutantofPhaC2
Ps
namedPhaC2
Ps
QKSTwasshownto
havehigherscl-mclPHAproductivityandalteredsub-
stratespecificity.WhenthismutatedPHAsynthasewas
expressedin
R.eutropha
PHB-4,therecombinantwas
reportedtoproduce42wt%scl-mclPHA[20].However,
theexpressionofthisenzymein
E.coli
wasnothigh
enoughforproducinglargeamountofPHA(Figure1).
Codonanalysisoftheoriginal
phaC2
Ps
QKST
gene
revealedthatalmost60%codonswerenotpreferredin
E.
coli
.Forexample,inatotalof23codonsforlysine,21
codonsareAAGwithafrequencyof27%inthe
E.coli
genome,whileonly2codonsareAAAwithafrequencyof
73%.VeryrarecodonssuchasAGG(Arg)andCUA(Leu)
alsoexist.Thus,codonoptimizationofthe
phaC2
Ps
QKST
genemayimproveproductivitybyincreasingproteinex-
pression.Therefore,PhaC2
Ps
QKSTwasoptimizedby
“
one
aminoacid-onecodon
”
strategy,resultinginnewPHA
synthasegene
phaC2
Ps
O
(Figure2).Also,theGCcontent
ofthecodingsequencewasadjustedfrom66.7%to58.3%.
Tofurtherenhancetheexpressionlevelofthetarget
protein,thehairpinstructurepHP17(Figure2),which
wasreportedtohavethelongesthalflifeamongahair-
pinmRNApool[29],waschosentoevaluateitseffect
ontheexpressionofPhaC2
Ps
QKST.Todothis,pHP17
wasinsertedtotheupstreamofthegene
phaC2
Ps
O
,
resultingin
phaC2
Ps
OH
[GenBank:JX082171].
EffectoftheoptimizationstrategiesonmRNA
degradationandproteinexpressionof
phaC2
Ps
Toassesstheeffectofcodonoptimizationandhairpin
stabilization,fivepET28a-basedplasmidswer