GB2518927A - The production of biomolecules using yeast - Google Patents

The production of biomolecules using yeast Download PDF

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GB2518927A
GB2518927A GB1408525.2A GB201408525A GB2518927A GB 2518927 A GB2518927 A GB 2518927A GB 201408525 A GB201408525 A GB 201408525A GB 2518927 A GB2518927 A GB 2518927A
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yeast
squalene
ethanol
growth
production
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Steven Kelly
Diane Kelly
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Swansea University
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Swansea University
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract

A method of producing squalene and ethanol using yeast, in particular Saccharomyces cerevisiae, is described. The squalene epoxidase gene ERG1 is placed under the control of a tetracycline-responsive promoter, and exposure to doxycycline is used to suppress gene expression and allow accumulation of squalene. The process is adapted for use with agricultural feedstocks, in one embodiment using a grass juice extracted from ryegrass.

Description

Intellectual Property Office Application No. GB1408525.2 RT\4 Date:2 February 2015 The following terms are registered trade marks and should be read as such wherever they occur in this document: Cheni Station (registered) Bioscreen C (registered) Intellectual Property Office is an operating name of the Patent Office www.ipo.govuk The PrSuction of Bithno.tec.ules Using Yeast Filed of the h.ventinn The. umsent invention. reiatcs Co the production of a. bmmqeciies. using yeast and in u ar nu not e i e the ooirn,ccu c s a stcroi or steiS pitt rsor uc.h as squaiene
Backgroundof the JnvefltiO
Squalene kthe ioopmie41 pttcuesor for a nUitber of steroki. Squatcue is a polunsau.t ated, tnterpem hs drocarbon (2,6,10,15 19,23 -haxamethyttctracosa 2,6JQF4,i.8.22hexaend) with nuUitional cornetic, p:harmaceutic.at ai4 ffiedieai applications The compound can be derived from plant oils and the hver oil & deep sea sharks, It has also been proposed that squalene is an important part of the MedIterranean diet and h it may have themoprotective properties, that can be. used to. protect an individual from eancert Squal'ene.ha also been used' in cosmetics to improve the naare of skm and u ha also been used an trnmunoIogcaI adjuant m accncs (riven the pctc:ntiai flr health hçnofits, there is an increasing. co.inrnercat demand fat sqUaene bitt this d.enand baa to be. hal need agtin.st conCerifi; abut planting crops w.pnxthve the compound as opposed to bod props nd also there arc cureotns Ibuthlarihe rahitats where.
squaJtne is ntracte from tsh The onçerns for the en tronment ncan4 tiLIt die ccht u squalene is high and also there is a need Itt ssir able ogrca of squaIene The present mvcition scck to OvrCrOfl tie peb ems ut the pm art L providine.
effective and sustainable way of producing hiomoiccules such as squaiene from plant materiak.
Summary of..the inve*itIon.
A.ceordin ta.:the. te5erit invention thete is prdyided a method of'podocing: a sqalene'hy fermentation of.a substnaa using a yeast..
Preferaby. the yeast is.Saeebaronwces and in particular Saceharomynes Cereviiae (., Cerev s ae lt* i euvisgedtl:t ii addtio to squ.ailene ethanol is prpduced..
Preferably the.S. Cerevisiae.is strain YUG37$3R0.i..
ft cri'v saged that FRGI gene tr<tnscnpuen \ inde tne costro1 of a doyc km -.
ftpttssbIe prQrnbtet hi parucufar the dox)urne-repressNe promoter is doxyevJinerepressbte fe/U 4JtCI ptomoter system Pretbnfbly.fermenttfflon i carried out at a low growth teniperature typically between 10 and.30 degrees. cenU grade, more typically between 15. and 25 degrees centigrade and even ntote articubtly betweeP 1:8 and 21 degrees centigrade It is further cnvisaged that.fernientation ía at a decreased oxygen availabuily.
it is prc*rcd thct the. 8Mb Watt for entatio.u i& g.nt8 As an alternatise t sutatrate ma' be tructai Preferably the grass ia hi the. form of grass jtiice and in partictdar the grass juice is an extract from rycgrass.
ft rt praered that the method nvcdvcs pLoducmg cpiaiene and nhnn by ernentaion of a ohmtbiomass.
it iS crvisagcd. ibti the platu hipt14$s is tn g1cLilturAfedstock Preferably the. tèedstock.1:s upj.. k.n:e.rited with doxyeyciine.. *1..
Desaiptinu f the Figures An emhothrnc.nt f he invention WiIi pow h described by Way ot example oply with reference to and. as iU.ustrated hi he aceonpanying Figqs in wtuch; flgure I shows:. The Ergoserol biosynthetic pathway hr yeast, Structures of squalene and sdeeed storol intermediates (boxed:); unbroken. arrow = single enzythatic. tep broken arrow = multiple enzymatic steps, Gene flares urper case, italicised.; protein names lower cae recuEar Figure 2 J'ows' Rdalie (%i abundance of ip ds tu VLG37 (opei baN and YCUI ERG] (filled bars) cultured using YPD mean VSes (h 2 [i S.Di. A squalene; B ergosterol; C aunt of all. i4aHdemethylated sterots; B sum oF l4cz'methyiated:StCrOiS.
Figure 3 ShOWS IJioscreen growth curves tbr YUQ37EkGJ at 2.0 C on. (3d t'ecdstock, f. ,I.. . . . . 1 I egcru inthuecc concentratufl o doxyc me (g n I), cuC\ Ibc S and 0 ag raE over1 Hgure $ shows.. A. GCMS.a&1a13srs of d4i3iERQL A) and B lotul ion ehtornatogn.ms io Y1JG;7ERU I gros.n on G I and on (U 50 dochne ml respeetiveiy.. J.x.: ergosterol: 2 ianos.tercu; 3 sqathene. C).and o):Frarnentauon, patterns for TMS den.'auzet1 rgosiuol and squaLnc cpectrvcv (MSI) Chemstattor NIs HI3P.AINIH Mass Spectrai.Ldirary Version 2.0).
Detailed Description. o:f the Invention
A he present un'entnn ieLatc tc the r,c of gcflCLCttE.) custoinised Saciharoiiiyces cerevisiae t produce eth:a.tiol. aud. additional tho-hnsea chenilcals. These.mate.rhtk are produced using sListMnahle.agrcndustrial. fcedsroc.ks (e.g... residual plant biom.ass) and process is of major interest to. the biofuel industiy q a way of producing costly material at ui cc. onornically visirle cost. The fl5vTt'n purticuIary relates a nticrobl.ai bion2finery Concept (it producing ethanot. and further, ethanol and squalene copro.duct:o.n using S. cereviv/ae ($rain YjG'FRni' Wherein ERG! (sqmul:eiie epn,xildase) transcription is undn the control of a doxycydline-repressible te&7-CYCI proüiotet. The product or ot ethanol and squalere In l 1G37-ERG gwwn u'ing ag wuUurau1ysourced grass juke suppktnetted with doxytyciine baa partic*u.IEm a vantages, over known Liroduction niethods.
Metb oth Yeast strain and growth media SqLaiene produeti i< sit.dw kcie i veer akw using a lihoravn strain of ccharornyecr cerer's'ac (FXJ37kRG1) in which seuakne epoxtdasc (LR&I protin,) cxprcssorf Is oqtrol ed using an opumsee uoxycyclincrcpressihc /tO/-T)'C/ promoter system The wdd4ype S cerevzstae parent strarn (VI G 37) w as ised as a comparator dining muial ccperuncnts Beth strains &c'e routhiely n'autaincc on oast neptore-dextrose (YPD) medium sontammg (ws) 2% g1uose, 2% there p&ptone and % yeast extract -ireluding 2% agar when required (aft media.eomponents supplied by Difeo).
For ethanol and squalene eoproduetkn expedments grass Juice (CU). feedstock was cxtritc.Ied from;ryefa5S..Lotn.un perenne supplied by the lnst.iUfl.e of BiologicaL Environmental Re search and Rural Scidnoes (II3ER.S. U.K). CU was. screened to remove i:irge partcubites, autodaved (T2P(.3.O. mm) and frozen $9 0) p.dor to use as a growth.
and Fermenut km substrate.
cn chro tography4nai spectrometry (GC-MS). pW.analysb CoB pek'ts. hom.expen.menud cuhurus wett taipunded in 7;.3 inethanoUwuter crUainin.g iS'3 1v potassium vdroruL 0 I i/y yy' wio ard C tg choleste el (as the neu'al st4ndazd\ and heated at 90 °C 1ot 2 ii Nonsapondiablc I ruth (quulcic and stc:ols) wcr extracted into class IIPLC vials using \ 2 nI hexane Ftiacts were evaporated to dryness using.a. centrifugal evaporator (tick Maxt dry plirs.) and deri:vatised using 100 1sL N &bis(trimethy1sUyi)frifluoroaeetamide, . and trimcthyichlorosflane (BSFFA-IMCS j99 11) ane 50 tiL auhdreus p)rul cc at Q C for 2 h (Martel ci ZUlCtaI, Triniethyisilyl iTMS)4eriyaud sthrOls, welre analyzed ing A 7890A.. GC*MS.
systeni. (Agilcn Technni,ogis) sihib a E.B-5MS. Utse,d silica ookthw (3d t O2 rntti 02S.pEt tiknlthiekness; JrW Scieriifl). The dv ttethpefatUt ras h1itiaUi hid at 79 °C for ii n then rn'reaed at 25 O( mm to a tina tempcarure cf 280 C. wh.icn was hdd par a. further 25 ruin. Samples wre.. snahed in s)l1tiess inede (1 pL inj.ee.tioti vóiuth u&ing helium earner, gas, electron impact ioñizatioki. (icir:t. source tm.eraturci of 1.50 cC) and scanning from. niiz 40 to 8.50.
CiC-MS dgta flies were anaJysed usng. MSP Enhanced ChemStariou software (Agilent Technolog cs lot, to deteunine squalenc and sterol,iiofiles for al iSo ates and lot oervat on of mtegrated peak aica erols were tuentified by rcktence to retention tunes and mass franeniaitor patterns for kno\n standards Phenot pie lipid anaIysi of strains In t,al experiments \eic undertaker to cetenune the clfcct of doxcy&ine on tie gwwth and lip d composition of the wild-tyie \ uci: 7 pat rot and aoxw cILne-repress' Pile \ UG3I4tRG/ s»=atn Smg e colories from each,ere used to inoculate 10 ml Dk4'nCs at YPD med'urn iontaning C-50 ug uockdlne niL 1 with staring cdl dci stues of 5 / mII' Cultures ere gr vti in 50 rnL flasks at 30 t 0 rpm for IS h af1cr hich time cell hiomass was.bat-vested by ecutrifugation Cdi pellets were dried. to constant mass fbr thornas (g d'-y eigh' 1 5 cktenmn IL..Ons and cJiulsi squ&enc via te'id conk it determmt d b' GC-MS i d%scnhed above P.roduetioe of ethanol andsqn.aknc from Ci fecds.toek.
Si!nidWiuo1u eo-pn$uciion Rxpe.nments ft achieve in1 lt:aaieous.c.u-produutiun of ethanol and qae.ne were perfôrmd in. I 00-wi.l IWnC.YCOITLb mi ropi$ea.u.ing.a. Rioscreen C (Dy. irowtli Curves i\b id F irdand) Iimfuin starting (th) t,ulture densit cs were acnieed by test spending a smgk Yl 037-f Pci! color in Cd and diktuig to obtain 5 < I 0' cbs nit in I ml of (11 oritain ig 0-50 tg doxvc ebne mL Start1ng Lultures sexe v rtexed mc abquotted soto Bioscreen wels (3 300 st rLphcaks per do c' cnn treatment) \I1 expert vents were mechged it 20 C t ypical cj ale production) in the thos'ccn (no 4takmg regime) 1or 96 h:, with optical density readieg (at: 600 nm) takmr. eve. ry 45. .nin Data as pottd ftopi.
the toscreen in ASCII format prior to aSyss. Dry weight determinations and GC-sMS lipid anthyses were pedormed on the; hioma.ss. fractions from pociled.Riosoreen. wells.
Growth parameters were derived with AOl) values. describe maximum 0.0.mhiimum On the Lg phrw N de°ned is if' c'ng h of tune a n iture spends n C n ot m..e\un no Of) T M ix aluc are cqawakni the time iahcn to reaU alf the maxim' n ccresc In growth of a culture v\Oj) 0.). Mini.tpqp (he., fastcst) douhlng times) fj were estm'tcd In dn idmg toe natural logar thir of 2 iy the fastest culture grcnth rates where p i. the gradient rifthe linear trend, line fitted to Iog4ransformcd 01) data.
Sequential produthon The stepwise production of ethanol and squiene was monitored wing the B.ioscreen.
YLG37-LRGJ aas first groxn far 48 h et 20 C umg QJ feeestoek, at t3h F3iocrth..n -nesuremcnts crt. suspenied rd 100 uf of supenatant re iosed train experuret tal wefls fir ethanol at £yras (\[arte & al 2010 This olwne was nwrc±atel) icplaecd with 100 ul of fresh (ii ccntamrng duxycycime (to g; e a fina' conerotranon of 5 or 50 ag doxcyJir mI) od the Bmerx,n rear1cd usr'g a tnedluni shaking egnne to pL'tnOte le;\ £° and squafene 4Lcuirulat on Di', weight dettxmiriatiou-. aur CC MS Ijaid anaiyses were performed on the b.i.ornass fractions hum pooled i3iosereeu. wells at tgah.
Sugw' cmd ethanol assays At. specific time. intervals (tdh:, t45hj.h. and tcah.Biosereen.meuuremeits wem suspended and. a 1 0...L veiume of culture supernarant removed:thmi repthsanuiti.ve expcrinwntal wells frtr ethanol. and uigar anulyms.. Sugar analyses1 were crfbr.med n @trtihl:y diluted Qypu a ly 200-fold) eulli i. rndwtn in 00 mM iotasium phcbsphale nFl 0, omaming tO mM. MgSO4, 1 mMNAD', 1.5 mM AlP And. 2,0 {J mL Leuesoc hwsen(etoWe>s glucnse&phrksphate dehydrugenac (Wirthn'gton Biodheaucal Cocpo& uon) Concenrations of glucose fructose, sucose and Nicta-' stere deerrrme 1 iron the dhanues j absci mice at 4O inn f6iiowin equential addition of 20 0 thL S. carey/s/ac 1-exokirae (Worthington Btodhemaal ( otporruon) 20 C inC C ech phesphoglucose isomerase, 1.5 [5 niLi S carey/s/ac sucrasefmaltase and 10 [5 ni[1 fruetanase.fr.mn Aspergilius niger respectively. Stantlarth o.f glueose4iuctose, sucrose and chicory maim wcrctscd to ealtbrarc the a;say.
Fthar& detcrrnnatiens werc maoc' ustng a &rcuraphotomctr cthanol asuy ku j< 1OH I bM6 Mewaiytre I td acLoramg to mariufaoture S rEstnKt ort AH samples verc i000Abid with distUied watr. pdorto an&ysi.&.
Reguiatku of grow b and qake atcnuIation Data from imual expermients ing \PD mectum &monstrates tnc potcnttf to rnaxlmLse squlene prnducuor from S cjevicwt throgi tgulatcin cf FROt cxpisIor T4bk I shoNc the plenotypic p d anaIy't of YE UP parent and LC37-FRGi muta-'t Pd! unures grown at 30 CC iSO rpm for 45 hon YPT) medwm Mean ajucs n = 2 k S Dl), IDOX doxycydilne. M:axhium sauajne contthtthd)1 A±e enThodened. dIal 1 4czdernethylated sceroi; r=*Èum, M i4c tneth)datd srdls.
Squ&ene 00)1 1 ipnl errnpocthrni (rng hpul g) Fatal Blotna& (jtg 14a-1'14q (mg Stram Ergosteio Sqna1ee (g L 1mg L 5 juL. JIM M 14S 033 0 6 33i 37c 050 O YUG37 W271 10031 327 91 10081 [0191 10351 103u YEJG3t-161 O9 040 528 455 76 3 08 j029] LAG! [3 °l [0071 10031 10241 1021 [0021 1W 020 or 4T 425 055 t025 YUGI7 3:19 1ø.31 161 W 061 O 13 jOOP ?02 0 311 YUG37 155 040 357 7 s8 430 1S04 * 43 r cm ERG! 10091 W0%] [(PD] 1°i' i021 I4 026 030 498)90 123 OAtS YUG37 3.03 [0.141 10221 10031 100I 10191 W 14 10u31 YUC37-37$ o'.-r' 424 55 25.0 luM 220J009: ERG! 1') 10! [0081 [0301 [0331 1Q28 [C 79] I 024 073 4i 395 107 US Yflfl7 1271 [0061 279[0 21 [0 361 10 301 1007] [0 14! \1G37 045 017 675 8% l75 ff93 I 58(0 12] FRG! O011 0321 10201 R1061 32II 1127 154 029 020 503 360 01 VEG3I 291 0 306 f04'J 101 0 111 0421 C 12 flG37 021 008 766 915 148 1130 L49fO.02] flG 0Ofl 41 R10fl 008] 0 UI 21 023 J4 463 3S 1.59 YIJC3' 93 0 6' 10231 [001! 1006! 02fl 1035! 0051 YVG37 0 1 0 22 7$ OdE 1 39 8' ERG! [0313] fO.0i.] 1.50 [0.03] JOM:] [0.10] [012] [0.93] TaMe.1 The Ergqs;ero.i hjc?.syrithdt.k pntbway in yea4 from SquaJçnQ 0 shown, in Figre; 1 nd the rs.dts 11x41.e* that, t1 rcpre$ioo of EkGI gene.e4çpe5sipn. mut be:sufficent tt rewft in nceased squ&ene ccurrat2tion but not compete growth nhrbztion inc highest squalene conrcjt (7 8 4 0 02 mg g' d biLmibs) aJ recorded m YUG37rRc? growr srng ypj) -uppkme' tec. v th SO ig d xyLychre rn however hccuse the hiomas ot tnee cultures was low (1,39 + 0d2 mg L'\ the. squlene yield was suboptimai (10.87 + 093.
rig i C1nvcrsel,, vnd't the squakn onte'rt ul Y1Xi3'ERGJ crown ti fl Qnç L3 doxycyclime m.Li was comparatively lower (357 0.2 mg g' dry tiomss), higher overa:tl cutuie grc.' Cf 3 028 ng)supporrec an mproei squknc yie d (Table 1, 1501 ± 42 rr I he re'u t Y iabe I shows that Doxyc.el it cd not alter hc hpic.
eontpos tron of the Y11637 patent acioss toe range of doMeveitte concentration (0 50 ug mL) tstcd Also there are no sigmtiant diflererces in the propo -non ol 1a-demmethiared or 1 4mtnethylated ster& intermediates were detected in treated \9J037 cultures sq shon m ligure 2 inc ii parncuta" thc s st-own m Figures 2C arid 21) which indicates that doxycydhne did not afkct the 1nction of other ERJ genes or proteins involved in ergosteroi biosynthesis. Changes in* the iiid composition of doxycycline treated YU637..ERC]J cultures, namely overall decreases in both I 4a-demnthytated and 4ttmethykmted.Sterdrj ipterniediate. are onsitcnt with the.spedflc inEiibition of qttaiCne epox.kilase expression.
thuwUi p nr3eLus 1w UG37 PRCi grown rig gr:ss iwt. (Ufl ac shc.wn in flIble 2 the growth paranieters fir YUG.374RG1 cultured on: cit 2.0 O( in the Bio sreen1⁄4 Me&n alue' n I I c D) ")OX doycyc me, -SoD rux nun -n umuur oncal density reding at $00 flot; Lag phase. ingth Of time culture remains at 10% of malumum On, I,2Ncax = time tiler' t' aehee half ma\nral cWtu'e ivr'vdi (n"rnnum Of) minimum liD x O: DTm fastest observed doubling time.
G oi th parametcr DOX.&g rnL 5 AOD Lag phue TAMn (h) (It) 0 L6 [0+Ofl fl.3.[O.4] 2LS [0.4J UfO.4j 0625 1 6([OOhl)6[0 4 io u05 163Oth1 115 04] fl12I 54(03] Ci 5$ 19+0.11 11:5 fft7 2W.9 [0:9] () [0-7] 0_S I 43[0J4 1l8[0] 3 +jC7] 860 I L34 {(L02j itS 0.4] 3SB [OAj Wi FOAl 3[001] ii5[07 38028] IO30']
Table. 2
Tahk 3 sbc'ws the GUcee frdose sucrose and iucan content ci (11 mcd a dunrg 1ernentaticn with \ 11037 / W?' Mean vaus r = 2 [-S Dl Super\cnpts 0 5 ai' reter w netha ontanng 05 eric 50 ig in dcxyt<ycun epeczne1y, Str kcthrrugh = net detected ii iiar Ti FiTr Thf'r Sugar Cheese 7.3 P Jfl 0.2 10,21 Fructose 212 2MJ 0.4 1-1] 0.5 0. U (L8 1:0.5] 0,6 FI Srcrose 0.5 [0+3] 0,2[0,2] 0,1 [0.2k t+2[0,2] 0J[O.Z] Fnataa i224] I7805 174 OO4 17812l 5[29] Totat 6i 6J PS 109 181102 19 141 6QI2l Tthfr 3:
U
Figure 3:tased on the inThrmatio.n from Tthht 3,. underscores data that highlight th potettial. for grass a a feedstocic for *S. cerevWae CU was rich in watr aoluhI carbohyrinitus as that. ite readily uti l.isS. tbr grtwi and than1 fonttez1ation, .Jn simultaneous. ethanol aM squrilene production *axparirnnts, *n$ximuln c:.eemrw:nr of ethanol ( Th.23 rug mLYYwerc. pi$1uce4 after 72 h of fernientetion,. Similar.ppenztatiqfl.5, 22 5 ft 0] rig ethanol v1 I) sere ecordec1 m sequential production exprirnents aer just 4 h, onor ts the addition ofnoxscschrc Taken as a whole, thee hanoI elds tn th study rc comparacie to those achteed pre icusly ui u (P and ahernaEn.e wild type laboratory straws of S Cerevciw The highe squa erie content C 89 i-025 rig g dry tuornass) and scualene >etds (180 418 trig L) work aJnevcd during snuttaneous prothiction experrn'ents ni v4i'ch (iT was upp1crrcnted (at uh) w ii' S and (1025 g do cyhne ML, repectnely Table 4 sos s Upid composition, dry weighi b omass and squalcrte sic1 of VI TGJ7 -ERrI itured using, Cii.. II cultures ntaintained at 20 C in the liescreen, Mean values (n = 2 ft S U), DOX doxycdhre Maxin'un squalene conten and yield arc emboldened Asterisks indicate secineinial production exedments supplemcn{od with additional.
() 4 DO)( after K' growth.. hi the absence of E)O( Strikethrougl.. not detect.ed..a sum.
of'nil I 4c!erncfth.yiacd.stdrols; sum of I 4ctmethyJated sterok Squidene BOX *Iipki. composition rrg lipk g") Total Biomas yied ___________---(irig g UM b1 4tM trgosten4 qualene (g Lt) tntg L I) 109 04' 525 <80 088 o en' [0 2J [0 16] 1 J ft 09] 0 3'S] [028' [039' fl97 027 398 7 8 18,0 jO4f'j o r 264 0401 [0t8] jO 35] 10 2] 073 1)28 S 4 S5 225 j02 3j 25< [021] C2fl [3J 031 009 592 895 178 263 [048] 0271 0 is' 011 [ ] Q01 139 928 l,4 L4 [09 [<9fl 27[03} L0c2M [ 11' O04 [070 1,34 945 1,45 11.4 [0 21] ft 0 [0 2O 222 655 540 120 --434')0i.
[0.28] [035] [044] [1.83] 24 625 2O 127 331jj937] jO2j [035 014 10241
Table 4
The highest total hiornass (52I4 g Ut) was recorded 48 Ii after therer oval of culture *supernatant and the addition of. fresh doxycychne-supplemented 63 (asterisked dath).
Jowever, concunutant with the acetimulation. of ergo.stetoi duxing. initiaL growth. in the absence of' ckm.yxy.cline (434 ± 10.07 end 3.80. ± 0.37 mi. trgostcrn4 g4 dry bi.oniae) quak e d' (12 0 1 1 83 end £2 7 024 g qui ene re ecukciy) wLre]owcr than thuce eco"ded ui suu1tancous nn)d cOny woicnncntc in whxh doxyc5o W3 wts nreseia ft:om t0[i. Tt use of bliorcactors to mairitaift optimal growth and 1drxunttion con.dition fr i.han& and squahne. coproducdon using Yi.JO37HERCU s now an. aenu thr 1 3* biorrocess deveiopment and commerciat scaieup. Using grass juice: fds:tock suppliemented with O5() pg doxyeyclioe rnL, Yt.JC 7-ERG.! fer.trerted. ethanol (22.5 [i 0 mg nd) il1ows fbi aecumuf anon of the highest squakoc eon cot (7 8' i 15 mg o div b mass ar d y icid (I g 0 4 8 i-rig squaho;e I I s itb n ppkmcn.s of 0 nd 0 0 ug doxycychne ml respretncly thass u cc wa fou'id to he nch n warc-soluble carbohydrater to! 1 r 3 rug sgrs irh) and pinirded excel cit Iccdsteck fbr growth and feirretation stadics using 1 G17_E/-?Gi I daL is onhnnu ii Figure 4 w're 0 s slio vu that that squateic proructron cai be anessed ni S cerejsae by repressing ERG! ge ic Lranscnption and hen. s the ponnal to co-produce etharol and sqafene (and/or addition] b'io-básed products) from, a single fcethtock. asthg yeast. Use of the rerG;-CYC promoter penritted rcgulauon of ERCI expression and squaknc aeuiirulttor iii t G3'?LJ?G I aPow cucurn enuon of the ietha grcwv.h phent.itype seen v her EfiGI is disrupted cormteb be potential for using the teJO7flCI piomoter vs cir to harres' s4ualene pmducriont on an' ind'us:flal scak is feasible beaus: dii' to the demand dnd bgh.
commercial value of squakne the costs of dxvcychne supplementation uulu likd) be offset:y those recoered troto squakoc production F udhcmiorc a is firportant that the promoter ci' choice: does nor have an cfIcr on yeast phvsio'lo'gi. a.asd so lids is the bcnoiir aNtic doxycychne sy.stem.
hi addition. to grass. because there E& shn,ultaneotìs saceharitication and ftnne,otation.
processes it is po'ssih:k for ftirthei.modi'fiedtion of Y1JG37FRGI to enable u'dlis'ation of fructan. which con. .rihanee the: effkienuy and,i'cir's of ethanol and squai'ene produced from gnlss juice.
It L5 aisci envisaged that the bubbtflile miy be any arhoLydrate bO cc tucu can be biokn down, thr exainpk by the ca hcdiydratv, may coi'e from a. variety of sotces sue i as ssastL from dan-v prc$ucvon *hich wil h'c lactose snars Other sources include carbohydrates from microbial origin or other carbohydrate sources that contain for example, lactose sucrose, glucose or a rr'xture of toese Tynical materials air h ororn ets from processes', such as industrial processes where there' is', a waste rn,ateri'a! left for exampte from hiomass production. food roeessi.n.g:or other agdcultural processes.
it is to he undEstodd ti:at the above embodiments have been provided only by w of cnipificnoon of this intitoa, suQh a those detailed bdiow. and that furthet thoclitk.atiotis and urtnroyemcns:tbcteto, as would be apparentt' peu'ons. skilled in the relevant art4 are deemed tq fall within th,e broad scope. anØ mbit of the prsent invertioo desenhed trthermnre where ?ndiyid m1 emnoduuers ae d sctssed tie unuitor1 b intended to avver comhinaüon.s of those emhnd.iment% ss.weft. The systen showt and described arenot lhnited.to the precise details and conditions cischysed. Method steps provided.niày not he hrit4 to the drderlin which the' are listed but may be ordered:any way as to can) ut he rn enuve pi ocess Uo 1 dcpamng fi err tw scope of dx in:vention, Furthermore, Other substitutions, hiodificatiørts, thange and emsssons,rna1y.be made in the design, oiierathic comlritbns aod attan ut&ts of rho exethplart ethbodiment without deporting froni the scopo of th invetitftm a expressed.
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CN110373338B (en) * 2019-08-15 2021-09-28 宜昌东阳光生化制药有限公司 Saccharomyces cerevisiae and application thereof

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