Posted on Authorea 2 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158316276.69268704 | This a preprint and has not been peer reviewed. Data may be preliminary. iest fti es eodisrglrprfloo at cd,ftyachl,biofuels alcohols, fatty acids, fatty catalytic of the portfolio explore to al., regular events et its metabolic Larroude many beyond M. evaluate and 2019; yeast genome Marx, this its & of modify Sauer, Alper, rapidly diversity & Egermeier, to Williams, 2017; us (Wagner, al., enabled mutagenesis (Celi´nska et 2019), transposon-based cloning Rossignol, 2019), Golden-gate & Xu, chromosomal and Nicaud, Xu, iterative & 2018) Trabelsi, Cre-LoxP-based & Zhou, editing, Larroude, genome Edwards, Holdridge, Macarena 2020) (Lv, collection Jin, Xu, 2020; integrations large & Engel, Hahn, Edwards, A (Yang, (Wong, & CRISPR-Cpf1 industry. assembly or Kim, nutraceutical 2020) gene Park, as and YaliBricks regarded (Bae, food ‘generally including CRISPR-Cas9 the a 2017), toolboxes, low- in as 2014) genetic upgrade recognized genetic al., customized been to of et also of opportunity ease (Groenewald has and tremendous the organism It us (GRAS) addition, compounds compounds. safe’ present In high-value lipid-related to growth damages. many feedstocks robust renewable membrane sequestrate value and lipophilic flexibility to with substrate environment associated manipulation, hydrophobic issues al., toxicity droplets a et oil the Zhou, provides of Larroude mitigate Koffas, compartmentalization bodies Macarena The Marsafari, lipid (Lv, NADPHs. 2017; and polyketides into al., HMG-CoA aromatic malonyl-CoA, et and acetyl-CoA, from 2019) (Gao derived Cao, carotenoids 2019) & Xu, 2016), Song, & Zhang, Stephanopoulos, oleochemicals (Jin, & 2017), terpenoids Stephanopoulos, Ahn, 2018), & Qiao, Xu, Zhou, Xu, Wasylenko, (Qiao, (P. lipids including compounds, lipophilic lipolytica production Yarrowia for factory cell oleaginous an as Introduction lipolytica late Y. the harness at to metabolism the baseline cell profiled a into chemicals. also as We reincorporated produced terpene-based serve mg/L). are or strain may (17.2 they squalene work strain engineered that of parental This observed the the and fermentation. to lipogenesis, level of compared mannitol ratio, from increase stage and C/N 28-fold competition acid the a citric flux optimizing flaks, byproducts By produced acetyl-CoA shake squalene metabolic respectively. strain in the for media, engineered squalene mitigating minimal host mg/L the acetate 502.7 and cycle, potential or about mannitol pH a glucose the media from as from squalene the NADPH lipolytica, led mg/L of controlling of reductase 188.2 Y. HMG-CoA recycling synthesis and yeast, native the mg/l scalable the oleaginous With 180.3 that and discovered about the improvement. and cost-efficient squalene of pathway highest the for engineering of the alternative squalene the bottleneck to the of promising reported identified Reconstitution a systematically we We as sustainable. work, traditionally production. not considered this is and Squalene is In cost-prohibitive thermostability. hosts and is squalene. compatibility microbial which lubri- skin extraction, in its super to plant pathway a due oil As biosynthetic industry or care pharmaceuticals. shark-hunting health steroids-based from in and widely sourced products used natural been triterpene-based has it for cant, molecule gateway the is Squalene Abstract 2020 5, May 2 1 Liu Huan in lipolytica production Yarrowia squalene for pathway mevalonate Optimizing ejn nvriyo hmclTechnologyUMBC Chemical of University Beijing 1 agWang Fang , tal. et sa nutiloegnu es hthsbe xesvl nierdt synthesize to engineered extensively been has that yeast oleaginous industrial an is h ioeet fti es ae taspro ott rdc hmcl htare that chemicals produce to host superior a it makes yeast this of lipogeneity The 1 iDeng Li , 1 n egXu Peng and , 1 2 tal et Recent . Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. / moimslae(im-lrc) .9gLCMLu(urs cec rdcs n.,4 / sodium g/L 0.825 41 (Difco), Inc.), sulfate) ammonium Products, 40 Science and (Sunrise acids ratio and CSM-Leu amino molar g/L (without (Difco), carbon/nitrogen Inc.) 0.69 base and sulfate) (Sigma-Aldrich), nitrogen Products, substrate sulfate ammonium yeast as ammonium Science g/L and g/L acetate 1.7 (Sunrise acids with sodium amino CSM-Leu made with was (without medium g/L 80:1 In fermentation base 0.69 YNB (Difco). nitrogen (Sigma-Aldrich), glucose. agar yeast sulfate g/L Bacto ratio g/L ammonium molar g/L glu- 1.7 g/L carbon/nitrogen g/L 15 with g/L and 5 with 1.1 made 20 substrate supplemented (Difco), as was and media glucose sulfate) medium 80:1 with YNB ammonium Inc.) of seed medium with and fermentation Products, YNB made YNB acids Science were process, amino plates (Sunrise fermentation glucose plates. 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HMG- Tang, (IPP) intermediate & of diphosphate (Xie reduction drugs sterol- the anti-cholesterol of through statins-related activity proceeds reactions, transcriptional trans- condensation the path- CoA acetyl-CoA (MVA) signal membrane controlling mevalonate with maintaining 2015), yeast-based and starts in The pathways, way Distefano, role 2001). defense & (Shimano, major plant (Palsuledesai (SREBPs) a of responsive-element-binding-proteins targeting play deployment more subcellular been Isoprenoids of the have for estimation duction, 2019). triterpenes An prenylation Pichler, and protein functions. & diterpenes biological homeostasis, (Moser sesquiterpenes, diverse nature monoterpenes, with from from products discovered ranging natural 2020) isoprenoids, of Alper, 70,000 group & metabolites than Nguyen, large secondary Miller, a 2019) Palmer, specialized al., are et 2019; more Isoprenoids (Jin al., access triterpenoids et to 2020), Marsafari, Xu, us (Lv, & allowed flavonoids (Marsafari and has sesquiterpenes including yeast values, this pharmaceutical in with effort engineering metabolic via M-o euts,wihi h aelmtn tpadtemlclrtre odsg many design to target molecular the and step rate-limiting the is which reductase, HMG-CoA NEB5 α arwalipolytica Yarrowia ihecec optn el eeotie rmNBfrpamdconstruction, plasmid for NEB from obtained were cells competent efficiency high μ /Lapcli a sdt cultivate to used was ampicillin g/mL o ffiin ytei fsuln rmsml ytei media. synthetic simple from squalene of synthesis efficient for .lipolytica Y. 2 idtp tanW9wsprhsdfo ATCC from purchased was W29 strain type wild .coli E. tan.Yatrc medium rich Yeast strains. tal et . Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. et ncmlac ihteYlBik ucoigpooo Wn ta. 07 og odig,Egl & Engel, compo- Holdridge, pathway Wong, 2017; previous al., containing et vector (Wong recipient protocol subcloning the YaliBricks into the enzyme with assembled compliance restriction and in the purified nents Genewiz, gel site by were KpnI verification fragments and sequence SnaBI the Upon at and 2017) the 2009). al., of al., downstream et into (Wong et DNA inserted backbone were genomic vector genes from pYLXP’ these primers the All the S2). with Table PCR-amplified and Zymoclean were Fast-S1 and genes Fisher Zyppy the from using All were purchased recoveries were research. of DNA Zymo enzymes gel from and restriction purchased clean-up, All PCR kits S1. miniprep, Table Plasmid supplementary enzymes. in Digest listed are primers All construction pathway and the Plasmid and with 2.4. min HPLC by 5 acid for acetic rpm curve and mL/min. 14,000 calibration mannitol, at Supelcogel the glucose, spec- centrifuged and of to UV-vis H was detector concentration according a broth index the (DCW) with fermentation refractive analyzing weight (OD600) The a for min. cell nm (g/L). used 4 600 dry 0.33:1 was for /min at to = supernatant holding 20 density converted OD600 and min, optical 260 be : 3 the to also DCW for measuring ramping could 175 /min by was that 20 monitored profile trophotometer then was temperature and growth column min, cell The 3 The for ml/min. holding 2 and of 200 (GC- velocity chromatography-FID to linear ramping gas a lipids to with vortexed of m injected was gas 7820A) (30 saponification directly mixture carrier (Agilent column system was complete The HP-5 (GC-FID) phase allow squalene. detector with hexane ionization extract to chromatography–flame equipped and to Gas vortex analysis. added min high-duty squalene was 10 for a hexane FID) for hydroxide with uL temperature temperature (sodium 400 room rpm room methoxide at Then 1,200 kept at sodium squalene. was at mixture of M 14,000 The hours extraction 0.5 pellet. at 2 and cell uL centrifugation the for by 500 resuspend shaking pelleted to and used was with withdrawn was subsequently methanol) completely and pure was in harvested Water dissolved was 5min. cell yeast for liquid rpm of units OD Four methods Analytical 2.3. 30 titer. and rpm 250 600 with tube. mL 50 fresh in single And 30 at 1997). grown Gaillardin, & Beckerich, (Chen, lipolytica the method into DNA/PEG transformed carrier were acetate/single-strand constructed plasmids pathway. of All C/N competing content biosynthesis) of the of acids effect (DMSO) except (fatty transformation dimethylsulfoxide the 80:1 precursor with explore Genetic ratio inhibit prepared to 2.2 respectively, to C/N solution g/L medium glucose-YNB in cerulenin fermentation 8.8 mg/L in them of into g/L, 1 components as pH added 4.4 And The same was g/L, The accumulation. as process. 2.2 squalenen were fermentation g/L, variation. on the 10:1 and 1.47 ratio pH in 20:1, 2012) to HCl indicate changed 40:1, al., M sulfate to 60:1, a et 0 ammonium ratio medium was (El-Ashgar 6. 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Data may be preliminary. aito fteegnsmyehnesuln ytei.A hw nFg B ooeepeso fylErg8, of co-overexpression 2B, Fig. in shown As synthesis. squalene enhance from may ylErg20 genes synthase. these and pyrophosphate of geranyl Erg12 variation encoding ylErg10, ylGPS kinase, ylErg8, synthetase; mevalonate pyrophosphate And encoding farnesyl Erg12 encoding encoding and thiolase, ylErg8 ylErg20 including acetoacetyl-CoA SQS and production, encoding squalene endogenous improve ylErg10 would pathway the kinase, MVA phosphomevalonate the of in genes overexpression synthesis. other squalene of the expression in to role critical addition a In plays domain membrane-binding N-terminal the that al., et The of (Gao 495bp region removed. truncated NADPH-binding the were an overexpressed of targeting) and then variation We (ER domain overexpressed. catalytic localization were the membrane 2017) containing for residues responsible C-terminal domain remaining N-terminal acid amino of form using truncated of endogenous potential When the system. strating yeast to translated (strain directly SQS be not could enzymes expressing strain from the co-expressed endoge- test (Fig. in we To with mg/L functions, produced production. was 83.76 better co-expressed squalene display at When for could production pomeroyi reductase beneficial squalene HMG-CoA Silicibacter was 2014). to of reductase led Jin, sources HMG-CoA (SctHMG) other & of whether HMG1 overexpression Kwak, truncated that the Thompson, indicating with 2A), 1998; strain the Lang, SQS, & nous in squalene Stahl, production a on isoprenoid (Polakowski, improving as HMG) ) in this by effective With be (encoded from to reductases proven tube. derived HMG-CoA test were three in HMGs of lipolytica (CSM-leu) three effect media The the synthetic investigated production. complete we chemically-defined strain, na- with by starting h ( accumulated 120 gene squalene at synthase no mg/L squalene almost endogenous was the there overexpressed addition, In (RODWELL, accumulation squalene tive 1976). in MITSCHELEN, step metabolic & rate-limiting NORDSTROM, the as reported Genome was reductase YALI0A10076g). HMG-CoA YALI0E05753g), (SQS1, synthase (Erg20, that squalene indicates pyrophos- Staring synthase and annotation mevalonate pyrophosphate (YALI0D17050g) YALI0E06193g), including synthase farnesyl 1). 8, pyrophosphate squalene, YALI0F05632g), geranyl (Erg synthesize (MVD1, ki- kinase to (Fig. decarboxylase mevalonate phosphomevalonate enzymes pathway (YALI0E04807g), YALI0B16038g), phate critical (MVA) reductase 12, HMG-CoA of mevalonate (Erg YALI0B08536g), number nase the 10, a (Erg from uses thiolase synthesized yeast acetoacetyl-CoA primarily condensation, acetyl-CoA was with squalene yeast, production In squalene for pathway mevalonate Debottlenecking PCR 3.1 for template was as discussions DNA used primers. were genomic and gene-specific samples and Results with genomic media gene the YPD integrated Then in the cultivated (Promega). of later kits verification was was genomic colony fragment Wizard screened genes linear with The functional The extracted screening. with Scientific). colony assembled Fisher for vector plate) Thermo pYLXP’ (Fermentas, into the NotI process, transformed into enzyme integration subsequently transformed restriction chromosomal were by In and linearized digestion was 1997). gel al., by et verified (Chen were plasmids assembled the All 2019). Xu, .lipolytica Y. .lipolytica Y. iiiatrpomeroyi Silicibacter ylHMG rnae omo M-o euts eodo -emnlmmrn agtn inlhsbeen has signal targeting membrane N-terminal of devoid reductase HMG-CoA of form Truncated . ylHMG HLYaliS01 otsri Po1g strain host xiisavreeeto ohsuln rdcinadcl rwh(i.2) indicating 2A), (Fig. growth cell and production squalene both on effect adverse exhibits u oteqikcnupino qaeeb ontemegseo ytae fe we After synthase. ergosterol downstream by squalene of consumption quick the to due ylHMG a mrv qaeesnhss otayt u yohss eoa fteN-terminal the of removal hypothesis, our to Contrary synthesis. squalene improve may .lipolytica Y. n endogenous and ,teegnee tanyedd113 gLsuln t10hi ettb,demon- tube, test in h 120 at squalene mg/L 121.31 yielded strain engineered the ), .lipolytica Y. eune(AIE40p,o hc h rt45ncetdsta noete165 the encode that nucleotides 495 first the which of (YALI0E04807p), sequence a ihyseicfrND Maose l,21)adti bacterial-derived this and 2016) al., et (Meadows NADH for specific highly was .lipolytica Y. otsri Po1g strain host Δ e sn h ihu ctt/igesrn are N/E method DNA/PEG carrier acetate/single-strand lithium the using Leu SpHMG otistecmlt eaoaeptwy(i.1.I V pathway, MVA In 1). (Fig. pathway mevalonate complete the contains ylHMG sapafr osnhsz aiu epns eas etdthe tested also We terpenes. various synthesize to platform a as hsrsl a ossetwt rvosfidnsta HMG that findings previous with consistent was result This . ihSS epciey o il fsuln 92 mg/L) (9.24 squalene of yield low A respectively. SQS, with .cerevisiae S. acaoye cerevisiae Saccharomyces Δ e n utvtdo S-e iia ei (agar media minimal CSM-Leu on cultivated and Leu 4 SQS acaoye cerevisiae Saccharomyces ,suln rdcinwsicesdt 17.25 to increased was production squalene ), eeas vrxrse ocmaehwthe how compare to overexpressed also were ylHMG ylHMG ee,w lotse hte the whether tested also we genes, (t495 , ylHMG ylHMG iiiatrpomeroyi Silicibacter a oepesdwith co-expressed was ocmaehwthe how compare to ) ecddby (encoded SpHMG SctHMG and from Y. Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. ys AL a anyue o upyo h yooi ctlCA hc a rvnt aetoisoforms two have to proven was which in acetyl-CoA, cytosolic to genes the route separate endoge- of two acetyl-CoA the supply by attempted primary for next encoded used the heterologous We mainly of is intermediate. was expression aldehyde which (ACL) the toxic lyase lyase, to the due citrate of impacted ATP accumulation negatively the nous was to fitness due growth possibly cell genes, the from that route observed We this from 3B). of (EcPuuc) efficiency dehydrogenase the acetylaldehyde compared and and squalene 2016) 1) (ALD) improve Maris, (Fig. dehydrogenase bypass , acetylaldehyde van could (ACS) (PDC), pathways & synthase decarboxylase pyruvate acetyl-CoA acetyl-CoA Pronk, the and Kozak, heterologous investigated we various Rossum, exam- First, van For and and production. 2016; endogenous yeast improve 2019). al., Bakers’ whether to Xu, et both investigated strategies & Meadows efficient in be Deng, 2019; production to Wang, Zhou, isoprenoid proven Marsafari, and were pathways precursor acids Liu, and critical acetyl-CoA fatty (Huan anabolism a cytosolic as lipid alternative both production found engineering in links was metabolite acetyl-CoA ple, It participated Cytosolic secondary pathway. metabolite pathways. MVA boost cy- intermediate in degradation to molecule Krebs acid the starting glycolysis, amino also the is and connecting is catabolism, oxidation intermediate Acetyl-CoA lipid after metabolic pathways. peroxisomal content essential lipid shunt synthesis, an and glyoxylate fitness is and cell acetyl-CoA, cle, increased NADPH, the to from ascribed Apart yield possibly decreased NADPH. is relatively of This despite supplement 3A). mg/L, the 135.22 (Fig. enhancing to DCW increased mg/g accu- titer 32.33 lipid production of during volumetric byproduct with major squalene ( more the ylHMG was and through mannitol SQS NADPHs why cytosolic with explain modulating partially in in could phase role re- mulation essential This more a an cycle. played Mannitol, of mannitol glucose, pathways production. basis to the squalene NADPH the compared improve cytosolic to alcohol on auxiliary results sugar fitness of best duced cellular the collection presented and a YALI0D18964g) production by tested encoded we squalene work, enhance this may In pathways co-expression Marsafari, these 1). Monireh how (Fig. Liu, investigated 2019) (Huan and al., (ylUGA2) pen- et dehydrogenase the Deng, semialdehyde dehy- on Li 6-phosphogluconate succinate relies ylMnDH2), and (ylMnDH1, primarily dehydrogenase (ylGND2) dehydrogenase NADPH originate isocitrate drogenase mannitol NADP-specific cytosolic may (ylMAE), include enzyme Minard sources, yeast pathways strain malic 1998; NADPH carbon Baker’s yeast (IDP2), McAlister-Henn, cytosolic as the & the Other Xuan, pathway. glucose of in phosphate Loftus, background With tose Jennings, NADPH genetic Minard, Lin, 2005). cytosolic and 2019; Wei, McAlister-Henn, source of Xu, (Cao, & carbon & source equivalent Deng, the work, Marsafari, on reducing Liu, previous depending as (Huan regulating routes on NADPH in alternative Based with various role A from critical reductively 2017). coenzyme plays is Hua, synthesis reductase (HMG-CoA) releasing and A HMG-CoA & pathway by acids coenzyme mevalonate mevalonate fatty 3-hydroxy-3-methylglutaryl 2015). the to 2019). in Stephanopoulos, in hydrolyzed al., precursor & et enzyme Ahn, (Ma rate-limiting rate-limiting Wasylenko, biosynthesis major first squalene 2017; the the al., is carbon- et as extend (HMGR) (Qiao reported and species stress also oxidative oleaginous was from in cell which protects equivalent backbones, reducing production carbon biological squalene primary improve the to as pathways NADPH precursor including acetyl-CoA nodes, and multiple carbon NAPDH at the Augmenting pathway repression. MVA improve that 3.2 transcriptional of further indicate SREBP-related regulation not results stringent or could These the inhibition pathway 36.24 to which feedback MVA SQS-ylHMG1. of due ergosterol-mediated in the expressing production possibly in strain only specific squalene, involved strain the toward a genes the for flux and the Among than obtained mg/L of lower was 107.08 gene. overexpression still production of sequential the is titer squalene of which with highest source DCW, overexpressed, the the mg/g were 2B), of SQS-ylHMG1 regardless and (Fig. synthesis, ylGPS combinations squalene these improve of further all not could Erg12 ylErg10, .cerevisiae S. SQS-ylHMG and E.coli .lipolytica Y. tanHLYaliS02 strain Fg B.B vrxrsino yuaedcroyae(cD)from (ScPDC) decarboxylase pyruvate of overexpression By 3B). (Fig. Fg A.Aogteecoe APs antldhdoeae(ylMnDH2, dehydrogenase mannitol NADPHs, chosen these Among 3A). (Fig. P u io tpaools 2017) Stephanopoulos, & Qiao, Xu, (P. .lipolytica Y. upyetr al 2,teegnee tanpoue 11% produced strain engineered the S2), Table Supplymentary , AL n C2 Nwosa,K (Nowrousian, ACL2) and (ACL1 E.coli 5 eotie ny165 gLo qaee(Fig. squalene of mg/L 106.54 only obtained we , .lipolytica Y. .lipolytica Y. . hnyMD2wsoverexpressed was ylMnDH2 When H i,Fn ag i & Li, Wang, Fan, Liu, (Hu eaoim T citrate ATP metabolism. c,Lsr Weltring, & Loser, uck, ¨ . hrfr,w next we Therefore, .cerevisiae S. .lipolytica Y. Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. u oteacmlto fcti cdwe lcs a tlzd h Hvrain eaieyaetstrain affect negatively synthesis, variations polyketide pH in The 3.5 utilized. to was 6 glucose from when pH two cultivation acid were of citric ratio declining of quick accumulation C/N for a the media competition observed to and we strong due pH work, a in cultivation previous growth to our Meanwhile, and leads In morphology production which 2017). cellular squalene lipid, al., affect of as that et level weight factors Xu similar critical cell (P. a acetyl-CoA 30%˜60% growth, precursor to cell the up ( for accumulating strains source producer, engineered carbon lipid our preferred in the detected optimization is was ratio carbon/nitrogen glucose and that pH imbalance with Despite metabolic strain factory. the engineered cell of to the cultivation due flask to Shake possibly effects 3.4 S2), burdensome causing Fig. overloading (Supplementary expression substrate gene as or acetate nor glucose neither ( strains engineered both When S1 D). strain Fig. S1 by squalene (Supplementary Fig. highest DCW (Supplementary mg/g the HLYaliS02 Strain productivity 29.04 strains, at highest engineered ). squalene the strain the of S3 with by Among yielding Table medium produced, flask. acetate-YNB and biomass shake in B g/L the h cultivate 6.6 in 168 to and pH at used depleted was the mg/L M) 191.68 adjust 0.5 reached to (HAc, titer used acid acetic was g/L to HCl 29.5 process M “shortcut” to conversion equivalently strains. the metabolic (NaAc), investigate engineered acetate a to the sodium explored strain Marsafari, as was g/L engineered Monireh strategy 41 serve the similar Liu, by S1). may a squalene (Huan study, acetate to this yield acetate In pathway that of and 2019). indicating al., efficiency g/L, et conversion Wang, 4.76 Fang carbon Stephanopou- improved to & Vogg, with up Qiao, acetyl-CoA Liu, polyketides Xu, J. work, produce cultivation 2017; another the al., to improvement when In et an bioreactors, (Qiao . to bench-top media 2017) acetate in led to los, g/L which pathway, media 115 glucose utilization to from g/L hexose switched 100 the was from to production (TAG) superior triacylglyceride even that of or equivalent reported & is been Dobrowolski, that has Rymowicz, pathway it Biegalska, Rakicka, example, Miro´nczuk, For 2016; Miro´nczuk, & 2017). Rymowicz, Mitu la, (Dobrowolski, production cals space squalene storage for the lipolytica media as Y. cell as serve of acetate also enhancement may and the content Glucose by squalene. cell. lipid 3.3 caused engineered of increased our was synthesis This in which content. The the squalene DCW lipid so 3B). for sequestrate mg/g increased ACL2 pathway (Fig. to MVA the 25.27 expressing mg/L the to by to 144.96 enrichment into due reduced acetyl-CoA growth squalene pushed yield for be of specific strategies could the titer pushing Surprisingly, acetyl-CoA the the strains cytosolic of to resulting adequate result leading the that a in ylHMG, probably obtained tes- and was was subsequently SQS increase synthesis were with squalene genes along in (YALI0D24431g) overexpressed increase ylACL2 19.5% and A (YALI0E34793g) ted. ylACL1 Endogenous 2000). ofrhripoesuln ytei,w sebe lC2t h lsi abrn Q,yHGand ylHMG SQS, harboring plasmid the ( to carbon strain ylACL2 as engineered assembled utilized the we But be production. synthesis, could ylMnDH2. terpene squalene acetate and improve and squalene (Supplemen- further glucose for To mg/L) value both (176.8 commercial that and acetate demonstrated application in data promising by squalene The a squalene F). of produced and amount to E similar sources S1 but in mg/L), engineered Fig. is (138.33 tary (which glucose cycle in mannitol the squalene that indicates This with Compared S3). Table C, and strain A S1 Fig. (Supplementary HLYaliS02 HLYaliS01 eecliae nguoeYBmdu,178 gLad181 gLsuln a achieved was squalene mg/L 188.18 and mg/L 157.81 medium, glucose-YNB in cultivated were ) a rwo ra ag fsbtae n ovr aiu rai at ohg-au chemi- high-value to waste organic various convert and substrates of range broad a on grow can HLYaliS01 with and SQS-ylHMG-ylMnDH2 nsitu In HLYaliS02 and HLYaliS02 .lipolytica Y. HLYaliS02 Hidctr(rmceo upe a sdt rc h Hcag n 6 and change pH the track to used was purple) (bromocresol indicator pH .lipolytica Y. ihayedo 65 ggDWad1.1m/ C,respectively DCW, mg/g 15.91 and DCW mg/g 16.53 of yield a with , HLYaliS04 rdcd102 gLsuln t10hi ctt-N medium acetate-YNB in h 140 at squalene mg/L 180.28 produced HLYaliS01 a eotdt ffiinl paeaei cda oecro source carbon sole as acid acetic uptake efficiently to reported was , HLYaliS03 n ctt saptniladcepidsra hmcl had chemicals industrial cheap and potential a as acetate and HLYaliS01 a ucinwl hnguoei sda abnsource. carbon as used is glucose when well function may ) intrsl na mrvdsuln rdcinfrom production squalene improved an in result didn’t ) and 6 tesri with strain (the HLYaliS02 , .lipolytica Y. HLYaliS02 .lipolytica Y. , HLYaliS03 HLYaliS01 SQS-ylHMG-ylACL2 and osse togaeaeutilization acetate strong possessed Sao 1999). (Szabo, HLYaliS03 ). ih9%o ctcacid acetic of 99% with , HLYaliS03 .lipolytica Y. SplmnayFig. (Supplementary HLYaliS01 rdcdless produced ) ihylACL2 with sanatural a is and Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. scniee sapoiigruet prd ua eeal edtc osuln.B reconstituting work, By this In squalene. squalene. to of amount feedstock considerable renewable achieved fermentation have sugar studies Microbial upgrade squalene of to few sustainable. of a not route source yeast, and Traditional promising in cost-prohibitive pathway a thermostability. mevalonate is as and extraction compatibility considered plant skin is oil with or lubricant shark-hunting super from a is compounds. terpene-related Squalene engineering and of potential squalene the of highlights Conclusion synthesis strain work efficient 4. the This for findings we to platform 2018). our strategies, level al., microbial with engineering squalene et promising consistent these similar Huang a was applying 2018; a as Choi, It By with & strain synthesis. 2013). Lee, yeast squalene Song, Vancura, (Seip, oleaginous nitrogen for & activity an and beneficial ACC1 Galdieri, obtained pathway was down have Zhang, slows synthase ratio and 2013; C/N acid kinase Hong, medium fatty Snf1 pathway that & activate of source to Zhu, Inhibition strategies malonyl-CoA phosphorylation He, effective the the kinase. Jackson, be through AMP to as controlled proven Snf1-mediated ACCase, was primarily by starvation cell was carboxylase residues to lipogenesis, production. acetyl-CoA serine during flowing squalene flux of pathway of carbon carbons improve downregulation sink of acetyl-CoA Further more to redistribution the lead the required squalene. and in may be of role provision important accumulation may (Supplementary an nitrogen the (ACC) production plays superfluous influenced squalene ratio C/N the strongly decreasing further that and was that with illustrated ratio obtained results speculated C/N These was We the growth. effect when However, adverse S4). ratio fermentation. 10:1, of observed C/N we Fig. or end form since the pathway, 20:1 DCW production MVA was at mg/g to to squalene g/L) flowed 32.6 titer the reduced and (1.9 to consumption, squalene enlarged than accumulation yield was highest acid higher the glucose flux citric with The 30.8% acetyl-CoA of h less that was productivity. 120 speculated which profile We at increased S3), fermentation mg/L media. Table with 502.75 80:1 supplementary similar reaching h Squalene B, 40:1, a 120 80:1. 4 ratio at C/N C/N 60:1, (Fig. for with mg/L at profile media 396 metabolic the set reached the in to achieved was 60:1 compared C/N ratio obtained, (Supplementary was at studied C/N acid was titer citric 80:1 the with weight, and supplemented cell 60:1 When dry 40:1, media mannitol, 20:1, YNB-PBS 10:1, S4). in including production ratios prevent squalene C/N Fig. Various may ACL). on B). by synthase ratio 4 cerulenin C/N (encoded acid (Fig. without of lyase fatty cerulenin media ATP-citrate effect endogenous by the YNB-PBS investigated the oxaloacetate the next and of in We acetyl-CoA inhibition h, that to 48 that than converted squalene at higher fact being the g/L was the from with 1.9 which citrate to reached obtained g/L, mannitol due was 9.7 Byproduct DCW possibly to S3). g/L supplemented, increased Table 14.9 result (Supplementary acid while acid the DCW citric citric utilized with mg/g but and was compared 25.78 mannitol, h glucose increase at consumption, 48 yield glucose 8.4% of at of an specific half work, profile media h, fermentation previous found: 188 YNB-PBS similar was our A at minimal A). accumulation mg/L with the 4 384.13 consistent to (Fig. to was cerulenin supplemented increased without which was production at process cerulenin squalene g/L the mg/L this 7.81 and 1 in reached synthesis, utilized acid Fig. squalene was PO citric (Supplementary enhance glucose of S3). and metabolism supplementation of Table cell hour into the (Supplementary ˜50% 48 reincorporated indicating DCW only at subsequently mg/g were But g/L The citrate 25.35 2.2 and S3). at strain to mannitol experiment. of yield up both biomass C) accumulated specific h; the S1 96 mannitol 6.0), squalene and byproduct control the pH Fig. pH major with (PBS, (Supplementary under The increase DCW solution fitness uncontrolled an g/L growth was buffer pH 13.98 better which from phosphoric S3), the reached results to M Fig. ascribed (Supplementary the 0.2 h is with improvement 168 with at compared media mg/L 88.4%, 354.44 YNB of to squalene minimal increased promote was to the production applied in squalene was strategy cultivated similar was a strain Thus, strain 2019). 1 active al., by with the et production Wang, buffer with Fang proton adduct PBS Marsafari, covalent combining of Monireh a by form Liu, loss irreversibly observed the of to was residue) known to (cysteine titer is site due polyketide Cerulenin transportation of supplementations. improvement nutrients cerulenin significant mg/L limit A and force. permeability driving membrane cell alter physiology, HLYaliS02 β- eoclAPsnhs,ihbtn h lnaino h at cdbcbn (Huan backbone acid fatty the of elongation the inhibiting synthase, ketoacyl-ACP soni i.4AadSplmnayFg 3.We h engineered the When S3). Fig. Supplementary and A 4 Fig. in (shown 4 3- ue a eaieyipc h lcs paert.T further To rate. uptake glucose the impact negatively may buffer 7 .cerevisiae S. .lipolytica Y. HLYaliS02 Hn Seo, (Han, Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. o-e hnfim moiie ihboorslprl Hsniieidctri rsneo surfactants. (2012). of A. presence S. Taya, in & indicator M., pH-sensitive T. El-Agez, purple M., bromocresol 2012 S. Chemistry, with Zourob, Analytical M., immobilized ISRN I. films wild- El-Nahhal, for I., thin strategy A. Sol-gel engineering El-Basioni, M., metabolic N. Gate-based El-Ashgar, Golden (2019). lipolytica. Yarrowia H. of crude Marx, strains & of type lipolytica. M., conversion Yarrowia Sauer, yeast Efficient M., by production Egermeier, oil (2016). cell yeast M. single dimorphic linalool Miro´nczuk, A. into the wastes & 207 of industrial Enhancing W., various transformation from Rymowicz, One-step glycerol P., Mitu (1997). la, A., C. Dobrowolski, Gaillardin, & (2017). M., J. lipolytica. Q. Beckerich, Yarrowia (2017). C., J.-M. D. Nicaud, lipolytica. Hua, Chen, Yarrowia & in C., manipulation pathway & Pauthenier, lipolytica. complex to 10 T., Biotechnology, dedicated Rossignol, system J.-Y., Yarrowia Assembly M., Gate Larroude, Golden R., Lin, yeast Ledesma-Amaro, Celi´nska, E., L.-J., oleaginous Wei, Base engineering doi:https://doi.org/10.1016/j.biortech.2017.06.105 Targeted by Sys- Disruption X., by CRISPR/Cas9 Gene the Multiplex with Cao, (2020). Combined Deaminase J.-S. Cytidine Hahn, Using & Genome B.-G., tem. lipolytica Kim, Yarrowia G., of B. Editing Park, S.-J., Bae, for Council Scholarship China the thank References like of to University like also would at would HL Engineering authors Environmental support. support. The funding and funding for Biochemical County National Chemical, project. Baltimore and of this Maryland OPP1188443) Department number supporting the (grant financially acknowledge Foundation to for Gates Melinda (CBET-1805139) & Foundation Bill Science acknowledge to like study. would this We of results the on based Acknowledgments filed been has patent provisional A manuscript. exper- the fermentation revised and interests PX engineering of genetic manuscript. Conflicts performed the HL wrote study. PX the and designed HL and iments. topic the conceived PX both profiled, serve contributions also may Author were work This level chemicals. fermentation. harness mannitol of terpene-based to and stage 40:1 or point acid late squalene mg/L C/N the starting citric 502.7 at with about byproduct a metabolism fermented produced cell as metabolic cerulenin into strain The mg/L reincorporated engineered were 1 flaks. byproducts The of shake supplementation ATP-citrate with in mode. conditions. or buffer squalene supply cultivation glucose PBS dehydrogenase acetyl-CoA with the from mannitol and conditioned optimized squalene reductase, NADPH media further mg/L HMG-CoA optimal we synthase, the 188.2 engineered identified squalene lyase, and the We of mg/l cycle, overexpression respectively. mannitol 180.3 Upon the media, about from minimal produced to NADPH acetate led overexpression) of or reductase recycling MnDH2 HMG-CoA the endogenous (with With that strain discovered improvement. and squalene reductase highest HMG-CoA the of number yeast, a oleaginous surveyed the We of engineering the reported we itcnlg ora,15 Journal, Biotechnology 237-243. , 2,4045 doi:10.1111/1751-7915.12605 450-455. (2), ple irbooyadBoehooy 48 Biotechnology, and Microbiology Applied 1,1028 doi:10.1002/biot.201900238 1900238. (1), .lipolytica Y. . ESMcoilg etr,366 Letters, Microbiology FEMS sa laiosyatfcoyfrcs-ffiin rdcinof production cost-efficient for factory yeast oleaginous an as .lipolytica Y. 8 irsu eho,245 Technol, Bioresour saptnilhs o qaeeproduction. squalene for host potential a as , 2,2225 doi:10.1007/s002530051043 232-235. (2), 4.doi:10.1093/femsle/fnz022 (4). irsu Technol, Bioresour 1641-1644. , Microbial Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. a .R,Wn,K-. ag .J,Dn,Y,Si .Q,Hag . i .J 21) dacsin Advances (2019). X.-J. Ji, & terpenoids. H., of Huang, production T.-Q., the Shi, for Facto- lipolytica Y., Cell Yarrowia Ding, Yeast of W.-J., engineering Oleaginous Wang, metabolic Optimizing the K.-F., Wang, (2019). Y.-R., P. Ma, Xu, & Biosynthesis. 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(3), oi irbooia 44 Microbiologica, Folia uoenJunlo ii cec n ehooy 113 Technology, and Science Lipid of Journal European Vl 4 p -4:Elsevier. 1-74): pp. 14, (Vol. 1) 5151.doi:10.1007/s00253-019-09892-y 5501-5516. (14), 1,5-2 doi:10.1021/cb500791f 51-62. (1), plMcoilBoeho,49 Biotechnol, Microbiol Appl 2) 7360-7370. (23), 10 eaoi niern omnctos 10 Communications, Engineering Metabolic 1,19-24. (1), β- xdto eitdstrategy. mediated oxidation 6,4942 doi:https://doi.org/10.1016/S0163- 439-452. (6), irsu eho,243 Technol, Bioresour a itcnl 35 Biotechnol, Nat aue 537 Nature, 1,66-71. (1), eaoi Engineering, Metabolic 393-399. , 1) 1299-1320. (11), 72) 694. (7622), 2,173-177. (2), e00121. , i-ACES, Biol J Biol J Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. ee r aiegnsfrom genes native are genes from decarboxylase pyruvate ScPDC1, 3 Fig. in pathway mevalonate geranyl 2 GPS, HMG-CoA kinase; Fig. synthetase; phosphomevalonate HMG, synthase. pyrophosphate 8, squalene carboxylase; farnesyl Erg SQS, 20, acetyl-CoA kinase; synthase; Erg mevalonate ACC1, pyrophosphate decarboxylase; 12, synthase; pyrophosphate Erg acetyl- mevalonate acid thiolase; ACS, MVD, acetoacetyl-CoA fatty complex; 10, FAS2, dehydrogenase Erg and pyruvate reductase; FAS1 pyru- PDH, PDC1, synthase; dehydrogenase; carboxylase; aldehyde CoA pyruvate isocitrate ALD, PYC1, NADP-specific cytosolic decarboxylase; dehydrogenase; IDP2, vate semialdehyde lyase; succinate citrate ATP UGA2, ACL2, dehydrogenase; enzyme; malic MAE1, hexokinase; HXK, 1 Fig. coenzyme acetyl regulates SNF1 mul- homolog captions AMPK Figures and yeast The acetylation. efficient histone (2013). and A. precise, Vancura, homeostasis & A CRISPR-Cas12a/Cpf1-assisted L., Galdieri, M., Zhang, (2020). 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C), (Supplement itcnlg ora,13 Journal, Biotechnology oprsno ieetHGCArdcaeadietfiaino aelmtn tp fendogenous of steps rate-limiting of identification and reductase HMG-CoA different of Comparison eaoi aha o qaeesnhssi laiosyat nH antldehydrogenase; mannitol MnDH, yeast. oleaginous in synthesis squalene for pathway Metabolic nacmn fNPHadaey-o rcro ahast mrv qaeeproduction. squalene improve to pathways precursor acetyl-CoA and NAPDH of Enhancement 3) 10848-10853. (39), E5308-E5316. (27), eaoi niern,36 Engineering, Metabolic .lipolytica Y. .lipolytica Y. 0426.diDI10.1128/AEM.02820-06 doi:DOI 2054-2060. , 5,1002 doi:10.1002/biot.201800022 1800022. (5), . p.1517.NwYr,N:Srne e York. New Springer NY: York, New 155-177). (pp. .cerevisiae S. oeua n ellrBooy 33 Biology, Cellular and Molecular n ealdgn noaincudb on nFg 1. Fig. in found be could annotation gene detailed and 99-115. , eaoi niern omnctos 10 Communications, Engineering Metabolic cuc leyedhdoeaefrom dehydrogenase aldehyde EcPuuc, ; 11 itcnlBon,114 Bioeng, Biotechnol rceig fteNtoa cdm of Academy National the of Proceedings eaoi niern Communications, Engineering Metabolic rceig fteNtoa cdm of Academy National the of Proceedings 2) 4701-4717. (23), 7,1521-1530. (7), irba Metabolic Microbial .coli E. ea n,30 Eng, Metab ple and Applied e00112. , Other . Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. i.2 Fig. 1 Fig. (B). 40:1 ratio C/N and cerulenin mg/L strain 1 with for Figures supplemented accumulation buffer, mannitol, squalene PBS consumption, and with glucose acid conditioned of profile media citric Fermentation acid weight, (A). cerulenin citric cell mg/L weight, dry 1 cell with dry supplemented and mannitol, strain buffer consumption, for glucose accumulation squalene of and profile Fermentation media. glucose-minimal 4 Fig. mrvn qaeepouto ycnrligp n / ai ihcrlnnsplmnainin supplementation cerulenin with ratio C/N and pH controlling by production squalene Improving HLYaliS02 utvtdi lcs-iia ei odtoe ihPBS with conditioned media glucose-minimal in cultivated 12 HLYaliS02 utvtdi glucose-minimal in cultivated Posted on Authorea 2 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158316276.69268704 — This a preprint and has not been peer reviewed. Data may be preliminary. i.4 Fig. 3 Fig. 13