FP2
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`|l||||IlllllllllllllllllllllllllIllllllllllllllllllllllllllllllllllIllllllllllllllHlllllllllll
`
`(10) International Publication Number
`
`(43) International Publication Date
`WO 2016/159869 A1
`6 October 2016 (06.10.2016) WlPOl PCT
`
`
`gg
`
`(51)
`
`International Patent Classification:
`C12P 7/64 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/SE2016/050274
`
`(22)
`
`International Filing Date:
`
`Filing Language:
`
`Publication Language:
`
`(74)
`
`(31)
`
`1 April 2016 (01.04.2016)
`
`English
`
`English
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(SE). YU, Tao; Hammarliden 8, S-412 62 Goteborg (SE).
`ZHOU, Yong-Jin; Uppstigen 121, S-412 80 Goteborg
`(SE). NIELSEN, Jens; Nedre Fogelbergsgatan 5, S—411 28
`Goteborg (SE).
`
`Agent: AROS PATENT AB; Box 1544,
`Uppsala (SE).
`
`S—751
`
`45
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(34)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, 82,
`
`Priority Data:
`62/142,236
`
`2 April 2015 (02.04.2015)
`
`US
`
`Applicant: BIOPETROLIA AB [SE/SE]; Chalmels
`Tekniska Hogskola, 3-412 96 Goteborg (SE).
`
`Inventors: DAVID, Florian; Eklandagatan 7, 8-412 55
`Goteborg (SE). SIEWERS, Verena; KVillegatan 29, 8-417
`08 Goteborg (SE). KRIVORUCHKO, Anastasia; Kjell-
`bergsgatan 5, S-411 32 Goteborg (SE). WENNING, Le-
`onie; Sagogfingen 37, Lgh 224, S-422 45 Hisingsbacka
`
`[Continued on next page]
`
`(54) Title: FUNGAL CELLS AND METHODS FOR PRODUCTION OF VERY LONG CHAIN FATTY ACID DERIVED
`PRODUCTS
`
`(57) Abstract: The present invention gen-
`erally relates to a genetically modified
`fungal cell capable of producing a very
`long chain fatty acid (VLCFA) and/or a
`VLCFA derivative. The genetically modi-
`fied fungal cell comprises at least one exo-
`genous gene encoding a fatty acyl-CoA re—
`ductase, and at least one gene encoding an
`elongase, and/or at least one gene encoding
`a fatty acid synthese.
`
`Figure 1
`
`Sugar
`
`”
`Acetyl»CoA
`flag“
`E]
`Malonyl‘CoA ”—1,
`“-
`Elongation sVstem forVLCFA
`i, EA§
`- ... .,.. mm w
`
`
`
`
`W W Fatty acylACoA _> W
`
`
`5qu [least]
`Bsdugases
`[C16:0/C18:0]
`FAS (Mycobaggzigl
`
`
`
`
`
`WEI New“
`.,
`.
`.. t
`..
`i U
` Elm-mg
`plant Woe)
`
`loin
`
`Fatty acyl-CaA
`
`(C16:1/C18:1)
`
`Eruizlc aclil(t122:lm9) <—
`
`Fatty acyl-COA
`(upteiulafion 010121)
`
`
`(cza;o(1)/c22:o(1))
`VLCFA reduggasgs
`MW
`Fatty alcohol
`
`(c20:o(1)/c22:o(1))
`mm
`mm
`VLCFA Wax esters
`“Jajbb‘a’ waik as???“
`(czn:o(1)/czz:a(1)/
`(CZD:1;C22:1;CZ4:1)
`
`C24:D[1))
`(uprazuladnn of men
`
`
`
`‘
`Docosanol (C22:0)
`(duwnreguladan oi Olel)
`
`
`
`
`
`
`wo2016/159869A1|lllllllllllllllllllllllllIlllllllllIllllllllllIlllllllllllll|llllllllllIllllllllllllllllllllll
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`

`

`WO 2016/159869 A1 |||||||||||l|lllIlllllllllllllll||||||l|||IIIIHIIIIIllllllllllllllIHIIlllllllllllllllllllllll
`
`-— ofinventors/zip (Rule 4.17(iv))
`$%'I‘JI\SI})’,ZEI\t/flioi?afi fiffififfig’gg: 2;: 2%,}: g: 11:11:
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, Published:
`LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE,
`SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17:
`
`— with international search report (Art. 21 (3))
`
`-- with sequence listing part ofdescription (Rule 5,2(a))
`
`— as to applicant’s entitlement to apply for and be granted
`a patent (Rule 4.1 7(iz'))
`
`

`

`WO 2016/159869
`
`PCT/SE2016/050274
`
`FUNGAL CELLS AND METHODS FOR PRODUCTION OF VERY LONG CHAIN FATTY
`
`ACID DERIVED PRODUCTS
`
`Field of the Invention
`
`The present invention relates to the development of genetically engineered fungal cells,
`
`preferably yeasts, that can produce specific chain length fatty acid derived products in a
`
`controllable and economic fashion. More specifically the invention relates to the production of
`
`very long chain fatty acid (VLCFA) products and derivatives, such as very long chain fatty
`
`alcohols, e.g., docosanol, very long chain fatty acids, e.g., erucic acid, nervonic acid, and wax
`
`esters of such very long chain fatty alcohols and fatty acids, e.g., jojoba oil esters, that can be
`
`used in the production of a range of industrial chemicals and oils, as well as pharmaceutical
`
`and cosmetic products.
`
`Background of the Invention
`
`Primary alcohols are a product class of compounds having a variety of industrial
`
`applications, this include a variety of biofuels and specialty chemicals. Primary alcohols also
`
`can be used to make a large number of additional industrial products including polymers and
`
`surfactants. Higher primary alcohols, also known as fatty alcohols, and their derivatives have
`
`numerous commercial applications,
`
`including use as surfactants,
`
`lubricants, plasticizers,
`
`solvents, emulsifiers, emollients, thickeners, flavors, fragrances, and fuels. Fatty alcohols can
`
`further be dehydrated to alpha-olefins, which have utility in the manufacture of polymers,
`
`lubricants, surfactants, plasticizers, and can also be used in fuel formulations.
`
`Current technologies for producing fatty alcohols involve inorganic catalyst-mediated
`
`reduction of fatty acids to the corresponding primary alcohols. The fatty acids used in this
`
`process are derived from natural sources, e.g., plant and animal oils and fats, primarily coconut,
`
`palm, palm kernel,
`
`tallow and lard. These various sources have different fatty acid
`
`compositions; of particular importance are the varying acyl chain lengths that are present. As
`
`a consequence, the fatty alcohols derived from these fatty acids also have varying chain lengths.
`
`The chain length of fatty alcohols greatly impacts the chemical and physical properties of the
`
`molecules, and thus different chain lengths are used for different applications. Fatty alcohols
`
`are currently produced from, for example, hydrogenation of fatty acids, hydroforrnylation of
`
`terminal olefins, partial oxidation of n-paraffins and the Al-catalyzed polymerization of
`
`ethylene. Fatty alcohols can also be made by chemical hydration of alpha-olefins produced
`
`from petrochemical feedstocks. Unfortunately, it is not commercially viable to produce fatty
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`alcohols directly from the oxidation of petrol eum-b ased linear hydrocarbons (n—paraffins). This
`
`impracticality is because the oxidation of n—paraffins produces primarily secondary alcohols,
`
`tertiary alcohols or ketones, or a mixture of these compounds, but does not produce high yields
`
`of fatty alcohols. Additionally, currently known methods for producing fatty alcohols suffer
`
`from the disadvantage that they are restricted to feedstock which is relatively expensive,
`
`notably ethylene, which is produced via the thermal cracking of petroleum. In addition, current
`
`methods require several steps, and several catalyst types.
`
`Plant primary fatty alcohols occur either in free form or are linked by an ester-bond with
`
`a fatty acid, e. g. palmitic acid, to give a wax ester or an aromatic compound, eg. femlic acid,
`
`to give an alkyl hydroxycinnamate. These various compounds are ofien components of plant
`
`extracellular lipid barriers: cuticle coating the aerial surfaces, suberin found in the cell walls of
`
`various internal and external tissue layers, and sporopollenin found in the outer walls of pollen
`
`grains. These waxes are usually complex mixtures ofvery-long-chain (Cm-€34)
`
`fatty acids and
`
`derivatives including primary fatty alcohols and wax esters. Wax esters can also serve as
`
`energy storage, such as in the case ofjojoba (Simmondsia chinensz's) seed oil.
`
`Unlike most other plants, the oil ofjojoba seeds, which constitutes between 45-55%, by
`
`weight, of the seeds, is mainly composed of very long chain monoesters of fatty acids and
`
`alcohols (97-98%, by weight) rather than triglycerides. These esters, which are commonly
`
`referred to as wax esters, are straight chain esters predominantly 36-46 carbons in length, with
`
`an ester bond approximately in the center of the chain. The oil, which exists as a liquid at room
`
`temperature, is used extensively as a raw material in the cosmetic and pharmaceutical industries
`
`for its dermatological properties. Jojoba oil is also used as an alternative to sperm oil as a
`
`lubricant and as a plasticizer. Because it is not subject to lipase hydrolysis and is thus poorly
`
`digested, jojoba oil has also been investigated as a non-caloric fat replacement in foods
`
`However, the relatively short supply ofjojoba oil and its extremely desirable properties
`
`have resulted in a rather high price, preventing its use for commercial preparation of a large
`
`number of useful derivatives and products.
`
`Thus, there exists a need for alternative means for cost effectively producing commercial
`
`and scalable quantities of very long chain length fatty acid derived products, including jojoba
`
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`oil.
`
`Previously, synthesis of long chain fatty alcohols and very long chain wax ester have
`
`only been demonstrated in yeast and Escherichia call? when heterologous expression of
`
`particular enzymes, including fatty acid reductase (FAR) and wax ester synthases, is combined
`
`with feeding of fatty acid substrates or relevant precursors (Kalscheuer et al., 2006; Li et al.,
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`2008; Teerawanichpan and Qiu, 2010). However, these solutions are not suitable for producing
`
`scalable quantities of very long chain fatty acid derived products in a cost—effective manner.
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`Summary of the Invention
`
`The present invention provides genetically engineered fungal cells, preferably yeasts,
`
`that include genes for the biosynthesis of very long chain fatty acid (VLCFA) products or
`
`derivatives, such as very long chain fatty acids, e.g., erucic acid, nervonic acid, very long chain
`
`fatty alcohols, e,g., docosanol, and/or wax esters, e. g. jojoba oil esters, and methods of
`
`producing such very long chain fatty acid products that can be used to produce a range of
`
`industrial chemicals and oils, e. g, lubricants, as well as pharmaceutical and cosmetic products,
`
`eg. emulsifiers, emollients, in a controllable and economic fashion,
`
`An aspect of the embodiments relates to genetically modified fungal cell capable of
`
`producing a VLCFA and/or a VLCFA derivative. The genetically modified fungal cell
`
`comprises at least one exogenous gene encoding a fatty acyl-CoA reductase and at least one
`
`gene encoding an elongase, and/or at least one gene encoding a fatty acid synthase, The at least
`
`one gene encoding the elongase is an overexpressed endogenous gene encoding the elongase
`
`and/or an exogenous gene encoding the elongase. Correspondingly, the at least one gene
`
`encoding the fatty acid synthase is an overexpressed endogenous gene encoding the fatty acid
`
`synthase and/or an exogenous gene encoding the fatty acid synthase.
`
`Another aspect of the embodiments relates to a genetically modified fiingal cell capable
`
`of producing a VLCFA or VLCFA derivative. The genetically fungal cell comprises at least
`
`one gene encoding a Mycobacterz’um fatty acid synthase.
`
`A further aspect of the embodiments relates to a method for the production of a VLCF A
`
`and/or a VLCFA derivative. The method comprises culturing a genetically modified fungal
`
`cell according to the embodiments in a culture medium. The method also comprises isolating
`
`the VLCFA and/or said VLCFA derivative from the genetically modified fungal cell and/or
`
`from the culture medium.
`
`The yeast Saccharomyces cerevisiae (S. cerevz'siae) is a very important cell factory as it
`
`is already widely used for production of biofuels, chemicals and pharmaceuticals, and there is
`
`therefore much interest in developing platform strains of this yeast that can be used for
`
`production of a whole range of different products. It is however a problem that such a platform
`
`cell factory for efficient production of fatty acid derived products is not as efficient as needed
`
`for good industrial application. This invention is,
`
`in an embodiment, a multiple gene
`
`modification approach of the yeast generating a stable and scalable platform for production of
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`PCT/SE2016/050274
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`very long chain fatty acid derived products by combining pathways for direct conversion from
`
`fatty acyl-CoA to long or very long chain fatty alcohols and/or wax esters.
`
`In one embodiment, the VLCFA derivative, e.g., fatty alcohols, fatty acid, wax esters
`
`etc, produced by the recombinant fiingal cell, such as yeast is a very long chain fatty alcohol,
`
`preferably docosanol which can be used for production of industrial chemicals or
`
`pharmaceutical and cosmetic products. In another embodiment, the VLCFA derivative is a very
`
`long chain fatty acid, preferably erucic acid ((Z)—docos-13-enoic acid) which is used as a
`
`component in industrial chemicals or pharmaceutical and cosmetic products. In yet another
`
`embodiment, the VLCFA derivative of this invention is nervonic acid ((Z)—Tetracos~15-enoic
`
`acid) which could be used in pharmaceutical and food products. For instance, nervonic acid
`
`can be used for the treatment of demyelinating diseases,
`
`including Multiple Sclerosis. In
`
`addition, nervonic acid can also be used for its nutritional value as a dietary supplement, for
`
`instance, in baby foods and/or infant formulas. In another embodiment, the VLCFA derivatives
`
`of this invention are wax esters, preferably jojoba oils/esters which can be used for production
`
`of industrial chemicals or pharmaceutical and cosmetic products. These and other aspects of
`
`the invention are set forth in more detail in the description of the invention below.
`
`Brief Description of the Drawings
`
`Figure 1. Shows synthesis of VLCFA, VLC-fatty alcohols and the corresponding wax
`
`esters. The background yeast strain (Apoxl, ACCJ **) provides enhanced precursor supply of
`
`malonyl-CoA for fatty acid elongation. The elongation towards very long chains was done via
`
`elongase or via fatty acid synthase (FAS) (Mycobacteria derived; evolved yeast FAS) systems.
`
`Heterologous very long chain specific reductases catalyze the reaction towards fatty alcohols.
`
`VLCFA wax ester synthases combine very long chain fatty acids with very long chain fatty
`
`alcohols producing very long chain wax esters. Depending on the product of interest the
`
`desaturase gene OLE] is upregulated (mono—unsaturated FAs) or downregulated (saturated
`
`FAs).
`
`Figure 2. Shows synthesis of wax esters, catalyzed by a fatty acyl-CoA reductase (FAR)
`
`and a wax synthase (WS).
`
`Figure 3. Shows fatty alcohol and wax ester biosynthesis. Gas chromatograms of shake
`
`flask cultures incubated for 48 hours in SD-URA + 2% glucose medium. The lines represent
`
`S. cerevisiae W03 strains that express Apis mellz'fera (Am) or Marinobacter aquaeolei VT8
`
`(MaFAlth) fatty acyl-CoA reductase (FAR) in combination with the wax synthase (WS)
`
`derived from Acinetobacter baylyi ADPl (Ab), Arabidopsz's thaliana (At), Euglena gracilis
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`(Eg) or Simmondsz'a chinensz's (Sc). The S. cerevz’sz’ae control strain, carrying the empty vector
`
`pSP—GM2, is also shown. The peaks highlighted by the grey bars labeled with I —— X were
`
`compared to NIST library standards and predicted to be: I, hexadecane (internal standard); II,
`
`palmitoleic acid (C1621) and palmitic acid (Cl6:0); III, oleic acid (C1811) and stearic acid
`
`(Cl8:0); IV, squalene; V, ergosterol; VI, hexadecanol (C1610); VII, octadecanol (C181) and
`
`octadecenol (C18: 1).
`
`Figure 4. Quantification of fatty alcohol in the producing strains described in Figure 3.
`
`Figure 5. Shows wax ester biosynthesis. Gas chromatograms of shake flask cultures
`
`incubated for 48 hours in SD—URA + 2% glucose medium. The lines represent S. cerevisiae
`
`JV03 strains that express Apis mellz'fera (Am) or Marinobacter aquaeolei VT8 (MaFAlth)
`
`fatty acyl-CoA reductase (FAR) in combination with the wax synthase (WS) derived from
`
`Acinetobacter baylyl' ADP]
`
`(Ab), Arabidopsis thaliana (At), Euglena gracilis (Eg) or
`
`Simmondsia chinensis (Sc). The S. cerevz'siae control strain, carrying the empty vector pSP-
`
`GM2, is also shown. The peaks highlighted by the grey bars labeled with I — II were compared
`
`to NIST library standards and were predicted to be: I, squalene and II, ergosterol. Peak III was
`
`identified by comparison of the mass spectrum to those published by Urbanova et al. 2012 and
`
`was identified as stearyl palmitate (C18:O-C16:0).
`
`Figure 6. Shows elongation of fatty acids up to C26 in the endoplasmic reticulum. The
`
`first step of the elongation process is catalyzed by a B—ketoacyl—COA synthase (KCS).
`
`Figure 7. Shows wax ester biosynthesis. Gas chromatograms of shake flask cultures
`
`incubated for 48 hours in Minimal medium + 2% glucose medium. The lines represent S.
`
`cerevz'sz'ae CEN.PK113-5D elo3AACC1** strains that express Apis mellifera (Am) fatty acyl—
`
`CoA reductase CFAR) in combination with the wax synthase (WS) derived from Acinez‘obacter
`
`baylyl' ADPl (Ab), Arabidopsis thaliana (At), Euglena gracilis (Eg) or Simmondsia chinensis
`
`(Sc). The S. cerevisiae control strain, carrying the empty vector pYX212, is also shown, The
`
`peaks highlighted by the grey bars labeled with I — VII were identified by comparison of the
`
`mass spectrum to those published by Urbanova et al. 2012 and were identified as I, palmityl
`
`myristate (Cl6:0 — C14:O); II, palmityl palmitate (C16:0 — C1620), stearyl myristate (C18:O —
`
`Cl4:0), palmityl palmitoleate (C16:O ~ C1621) and stearyl myristoleate (C1820 — C14zl); III,
`
`stearyl palmitate (C18:0 — Cl6:0); arachidyl myristate (C20:O — C14:O), stearyl palmitoleate
`
`(C1820 — Cl6:1), palmityl oleate (Cl6:0 — C18zl) and oleyl palmitoleate (C18:l — C16:1); IV,
`
`arachidyl palmitate (C20:0 — Cl6:0), behenyl myristate (C22:0 — C1420), palmityl arachidate
`
`(Cl6:l — C2020), stearyl stearate (C1810 — C1820) and arachidyl palmitoleate (C20:0 — C16: 1);
`
`V, behenyl palmitate (C22:0 — C16:O), palmityl behenate (Cl6:0 — C22:0), arachidyl stearate
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`(C20:0 — C18:0), stearyl arachidate (C1820 — C20:0), behenyl palmitoleate (C22:0 — C16: 1)
`
`and arachidyl oleate (C2020 — C1821); VI, behenyl stearate (C2220 — C1820), arachidyl
`
`arachidate (C2020 ~ C20:0), stearyl behenate (C1820 — C2220) and behenyl oleate (C2220 —
`
`C1821); VII, arachidyl behenate (C20:0 — C2220) and behenyl arachidate (C2220 — C2020).
`
`Figure 8. Shows wax ester biosynthesis. Gas chromatograms of shake flask cultures
`
`incubated for 48 hours in Minimal medium + 2% glucose medium The lines represent S.
`
`cerevisiae CEN.PK113-5D elo3A ACC1** strains that express Marinobacter aquaeolez' VT8
`
`(MaFAlth) fatty acyl-CoA reductase (FAR) in combination with the wax synthase (WS)
`
`derived from Arabidopsz’s thaliana (At), Euglena gmcilis (Eg) or Simmondsia chinensis (Sc).
`
`The S. cerevz’siae control strain, carrying the empty vector pYX212, is also shown. The peaks
`
`highlighted by the grey bars labeled with I —— VII were identified by comparison of the mass
`
`spectrum to those published by Urbanova et a1. 2012 and were identified as I, palmity1 myristate
`
`(C1620 — C14:0) and palmitoleyl myristate (C16:1 — C1420); II, palmity1 palmitate (C16:0 —
`
`C16:0), stearyl myristate (C18:0 — C1420), palmity1 palmitoleate (C16:O — C1621), stearyl
`
`myristoleate (C1820 — C1421) and palmitoleyl palmitoleate (C16:1 — C1621); III, stearyl
`
`palmitate (C1820 — C16:0), palmity1 stearate (C1620 — C18:0), arachidyl myristate (02020 —
`
`C1420), stearyl palmitoleate (C1820 — C1621), palmity1 oleate (C1620 —— C1821), oleyl
`
`palmitoleate (C1821 — C16: 1) and palmitoleyl oleate (C16:1 — C18: 1); IV, arachidyl palmitate
`
`(C2020 ~ C16:0), behenyl myristate (C2220 — C1420), palmity1 arachidate (C1620 — C2020),
`
`stearyl stearate (C18:O — C1820) and arachidyl palmitoleate (C2020 — C16:1); V, behenyl
`
`palmitate (C2220 — C16:0), palmity1 behenate (C1620 — C2220), arachidyl stearate (C2020 —
`
`C1820), stearyl arachidate (C1820 — C20:0), behenyl palmitoleate (C2220 — C1621) and
`
`arachidyl oleate (C2020 -— C1821); VI, behenyl stearate (C2220 — C18:0), arachidyl arachidate
`
`(C2020 — C20:0), stearyl behenate (C1820 — C2220) and behenyl oleate (C2220 — C1821); V11,
`
`arachidyl behenate (C2020 — C2220) and behenyl arachidate (C2220 — C2010).
`
`Figure 9. Quantification of fatty alcohol in the producing strains described in Figure 7
`
`and 8.
`
`and 8.
`
`Figure 10. Quantification of wax esters in the producing strains described in Figure 7
`
`Figure 11. Shows the concentration of C3 8 to C42 wax esters in strain 5De103AACC1 **
`
`(p YXZ 12: 2MaFA lth.‘ :Sci WS: :EloZ).
`
`Figure 12. Shows the specific m/z peaks of C40 wax esters in strain 5Delo3AACC1**
`
`(pYX212: MaFA lth::Sci WS: :Elo2).
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`Figure 13. Shows the specific m/z peaks of C42 wax esters in strain 5D6103AACC1**
`
`(pYX212: MaFAlth::Scz'WS::Elo2).
`
`Figure 14. Shows docosanol production in one independent clone of a control strain
`
`(JV03 Aelo3 pEL02) and producing strains (W03 Aelo3 pEL02 pAtSFAR)
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`Figure 15. Shows a schematic illustration of the genome engineering strategy for
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`integrating ELOl, EL02 and AtSFAR overexpression, at the same time as deleting ELO3.
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`Figure 16. Shows docosanol production (mg/L) when comparing the two strains W03
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`(Ae103 pEL02 pAtSFAR) and CENPK ll3-5D (Aelo3Agall GAL7p-ACC1** GAL7p—
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`AtSFAR GAL7p-ELOl GALlOp—ELOZ).
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`Figure 17. Shows VLC fatty acid synthesis in S cerevz‘sz‘ae through overexpression of
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`elongase genes EL02, AtFAEl (Arabidopsis thaliana), BnKCS (Brassica napus), CaKCS
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`(Crambe abyssinica), LaKCS (Lunarl‘a annual), ScFAE (Simmondsia chinensis), TmKCS
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`(Tropaeolum majus) in combination with overexpression of S. cerevisiae derived desaturase
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`OLEl in the background strain CENPK 113-5D Aelo3 ACC1**.
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`Figure 18. Shows overexpression of S. cerevz'sz’ae gene OLE] in combination with EL02
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`and its effect on increasing mono unsaturated fatty acid levels. As a background strain CEN.PK
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`ll3-5D Aelo3 ACC1** was used.
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`Figure 19. Shows VLC fatty acid CoA biosynthesis. Gas chromatograms of shake flask
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`cultures incubated for 72 hours in SD-LEU + 2% glucose medium. The lines represent S.
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`cerevz’sz’ae TDY7005 strains that express p415GPD::MVFAS::Acps. The S. cerevisiae
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`TDY7005 control strain is also shown. The peaks highlighted by the grey bars labeled with I —
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`X were compared to NIST library standards and predicted to be: I, Heneicosylic acid (internal
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`standard, C21 :0);
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`II, Behenic acid (C22:0);
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`III, Tetracosanoic acid(C24:O) and IV,
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`Hexacosanoic Acid (C2620).
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`Figure 20. Shows C22 fatty acid production from two different systems. Comparing the
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`production in JVO3 Aelo3 pEL02 and TDY7005 Aelo3Ae102 p415GPD::MvFAS::Acps.
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`Figure 21. Shows production of erucic acid in S. cerevisiae. Overexpression of S.
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`cerevisiae derived desaturase OLEl and specific elongases in strain background CENPK 113-
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`5D Aelo3 ACC1**. Elongases include EL02 (S. cerevisiae), AtFAEl (Arabidopsis thaliana),
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`BnKCS (Brassica napus), CaKCS (Crambe abyssinica), LaKCS (Lunaria annua) and ScFAE
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`(Simmondsia chinensis).
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`Figure 22. Shows the metabolic pathways for production of fatty alcohols (i) directly
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`from fatty acyl—CoA or (ii) via free fatty acids and fatty aldehydes.
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`Figure 23. Shows the quantification of fatty alcohols directly synthesized from (i) fatty
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`acyl—CoA or (ii) via free fatty acids and fatty aldehydes. The fatty alcohols were extracted from
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`48-h shake flask cultures in glucose minimal medium as previously described (Buijs et al.,
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`2015). F aCoAR represents the Strain YIZOl (MA Ta A/[ALZ—8c SUCZ 111193211 ura3—52 hdeA)
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`(Buijs et a1., 2015) and expresses FAR from Marinobacter aquaeolei (Willis et al., 2011), and
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`CAR represents YJZOl and expresses MmCAR from Mycobacterium marinum (Akhtar et al.,
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`2013). The corresponding co-factor phosphopantetheinyl transferase NpgA from Aspergillus
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`nidulans was also overexpressed (Mootz et a1, 2002).
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`Figure 24. Show the overexpression of endogenous yeast genes MPP6, ACP], EPT],
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`FAA], GEP4, GGAZ, IDP3, [NP54, LPP], MCRJ, ORM], RTC3, SP07, TGL1, YFT2 using
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`plasmid pSP—GM212AmFAR (SEQ ID NO: 1, Partow et a1. 2010) in yeast strains CEN.PK 113—
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`5D and JVO3. Relative quantification of fatty alcohol (C1821) profiles compared to the
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`particular control strain carrying pSP-GM222AmFAR without any coexpression.
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`Figure 25. Shows alcohol biosynthesis in Y.
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`lipolytz’ca. Gas chromatograms of shake
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`flask cultures incubated for 48 hours in SD-URA-LEU + 2% glucose medium. The upper graph
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`shows the Y. Zipolytica JMY195 background strain, the lower strain shows the same strain
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`expressing the Apis mellzfera (Am) fatty acyl—CoA reductase (FAR) in combination with the
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`wax synthase (WS) derived from Simmondsia chinensis (Sc). The peaks highlighted by the
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`grey bars labeled with I — II were compared to NIST library standards and predicted to be: I,
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`hexadecanol (C16:O) and II, octadecanol (C18: 1).
`
`Detailed Description of the Invention
`
`The present
`
`invention now will be described hereinafter with reference to the
`
`accompanying drawings and examples, in which embodiments of the invention are shown. This
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`description is not intended to be a detailed catalog of all the different ways in which the
`
`invention may be implemented, or all the features that may be added to the instant invention.
`
`For example, features illustrated with respect to one embodiment may be incorporated into
`
`other embodiments, and features illustrated with respect to a particular embodiment may be
`
`deleted from that embodiment. Thus, the invention contemplates that in some embodiments of
`
`the invention, any feature or combination of features set forth herein can be excluded or
`
`omitted. In addition, numerous variations and additions to the various embodiments suggested
`
`herein will be apparent to those skilled in the art in light of the instant disclosure, which do not
`
`depart from the instant invention. Hence, the following descriptions are intended to illustrate
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`some particular embodiments ofthe invention, and not to exhaustively specify all permutations,
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`combinations and variations thereof.
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`Definitions
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`Unless otherwise defined, all technical and scientific terms used herein have the same
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`meaning as commonly understood by one of ordinary skill in the art to which this invention
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`belongs. As such, the elongases, reductases, desaturases, fatty acid synthases and wax ester
`
`synthases, polypetides and genes encoding them, that may be used in this invention are any of
`
`those known in the art or homologues or derivatives thereof.
`
`The terminology used in the description of the invention herein is for the purpose of
`
`describing particular embodiments only and is not intended to be limiting of the invention.
`
`All publications, patent applications, patents and other references cited herein are
`
`incorporated by reference in their entireties for the teachings relevant to the sentence and/or
`
`paragraph in which the reference is presented.
`
`Unless the context indicates otherwise, it is specifically intended that the various features
`
`of the invention described herein can be used in any combination. Moreover, the present
`
`invention also contemplates that in some embodiments of the invention, any feature or
`
`combination of features set forth herein can be excluded or omitted. To illustrate, if the
`
`specification states that a composition comprises components A, B and C, it is specifically
`
`intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed
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`singularly or in any combination.
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`To facilitate understanding of the invention, a number of terms are defined below.
`
`As used in the description of the invention and the appended claims, the singular forms
`
`“a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly
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`25
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`indicates otherwise.
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`Also as used herein, “and/or” refers to and encompasses any and all possible
`
`combinations of one or more of the associated listed items, as well as the lack of combinations
`
`when interpreted in the alternative “or”).
`7) (C
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`The term “comprise,
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`comprises” and “comprising” as used herein, specify the presence
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`30
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`of the stated features, integers, steps, operations, elements, and/or components, but do not
`
`preclude the presence or addition of one or more other features, integers, steps, operations,
`
`elements, components, and/or groups thereof.
`
`As used herein, the transitional phrase “consisting essentially of’ means that the scope
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`of a claim is to be interpreted to encompass the specified materials or steps recited in the claim
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`and those that do not materially affect the basic and novel characteristic(s) of the claimed
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`invention. Thus, the term “consisting essentially of” when used in a claim of this invention is
`
`not intended to be interpreted to be equivalent to “comprising”
`
`As used herein the term “recombinant” when used means that a particular nucleic acid
`
`(DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation
`
`steps resulting in a construct having a structural coding or non-coding sequence distinguishable
`
`from endogenous nucleic acids found in natural systems.
`
`As used herein, the terms “protein” and “polypeptide” refer to compounds comprising
`
`amino acids joined via peptide bonds and are used interchangeably.
`23 u-
`a) (4'
`1ncreases,
`increased,” “increasing,
`
`As used herein, the terms “increase,
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`’7 KC
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`10
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`enhance,”
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`“enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof) indicate an
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`elevation of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,
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`30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%,
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`200%, 300%, 400%, 500% or more, or any range therein, as compared to a control.
`3’
`(C
`,3
`(6
`
`As used herein,
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`the terms “reduce,
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`reduces,
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`reduced,” “reduction,” “diminish,”
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`“suppress,” and “decrease” and similar terms mean a decrease of at least about about 0%, 1%,
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`2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
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`60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or
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`more, or any range therein, as compared to a control.
`
`A reduced expression of a gene as used herein involves a gentical modification that
`
`reduces the transcription of the gene, reduces the translation of the mRNA transcribed from the
`
`gene and/or reduces post-translational processing of the protein translated from the mRNA.
`
`Such genetical modification includes insertion(s), deletion(s), replacement(s) or mutation(s)
`
`applied to the control sequence, such as a promoter and enhancer, of the gene. For instance,
`
`the promoter of the gene could be replaced by a less active or inducible promoter to thereby
`
`result in a reduced transcription of the gene. Also a knock-out of the promoter would result in
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`reduced, typically zero, expression of the gene.
`
`As used herein the terms “knock-out” or “deletion” or “disruption” refers to a gene that
`
`is inoperative or knocked out and/or a nonfunctional gene product, eg. a polypeptide having
`
`essentially no activity, eg. less than about 10% or even 5% as compared to the activity of the
`
`wild type polypeptide.
`
`As used herein,
`
`the term “portion” or “fragment” of a nucleotide sequence of the
`
`invention will be understood to mean a nucleotide sequence of reduced length relative to a
`
`reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or
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`consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical, e. g.
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`80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
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`96%, 98%, 99% identical, to the reference nucleic acid or nucleotide sequence. Such a nucleic
`
`acid fragment or portion according to the invention may be, where appropriate, included in a
`
`larger polynucleotide of which it is a constituent.
`
`Different nucl