(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization _
`International Bureau
`
`' |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`
`
`(43) International Publication Date
`
`20 September 2007 (20.09.2007)
`
`(51) International Patent Classification:
`CIZN 1/18 (2006.01)
`
`(10) International Publication Number
`
`WO 2007/106524 A2
`
`Kari [Fl/Fl]; Makitorpantie 17 D 39, FI—00640 Helsinki
`(Fl). HAUSE, Benjamin, Matthew [US/US]; 1575 250th
`Avenue, Currie, MN 56123 (US). MCMULLIN, Thomas,
`William [US/US]; 3509 Lilac Lane, Minnetonka, MN
`55345 (US). ROBERG-PEREZ, Kevin [US/US]; 2250
`Cleveland Street, Minneapolis, MN 55418 (US).
`
`Agent: COHN, Gary, C.; Gary C. Cohn PLLC, 1147 N.
`Fourth Street, Unit 6E, Philadelphia, PA 19123 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS,
`JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS,
`LT, LU, LY, MA, MD, MG, MK, MN, MW, MX, MY, MZ,
`NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU,
`SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(21) International Application Number:
`PCT/US2007/006408
`
`(22) International Filing Date:
`
`13 March 2007 (13.03.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(74)
`
`(81)
`
`(30) Priority Data:
`60/781,674
`
`13 March 2006 (13.03.2006)
`
`US
`
`(71) Applicant (for all designated States except US): NATURE
`WORKS LLC [US/US]; 15305 Minnetonka Boulevard,
`Minneapolis, MN 55345 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): DUNDON, Cather-
`ine, Asleson [US/US]; 3944 45th Avenue S., Minneapolis,
`MN 55406 (US). SUOMlNEN, Pirkko [Fl/US]; 7801
`Kingsview, Maple Grove, MN 55311 (US). ARISTIDOU,
`Aristos [CY/US]; 7809 Kingsview, Maple Grove, MN
`55311 (US). RUSH, Brian, J. [US/US]; 3336 Emerson Av—
`enue S, Minneapolis, MN 55408 (US). KOIVURANTA,
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`
`[Continued on next page]
`
`
`
`07/106524A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`(54) Title: YEAST CELLS HAVING DISRUPTED PATHWAY FROM DIHYDROXYACETONE PHOSPHATE TO GLYCEROL
`
`pBH165
`6846 bps
`
`\
`
`
`
`
`% K K1tbrrnlogous Flank x0”
`
`
`
`EcoRl
`
`,
`
`3'GPD1
`
`
`
`
`
`l
`5‘5
`Xnal N ...-
`
`
`c (57) Abstract: Yeast cells are genetically modified to disrupt a native metabolic pathway from dihydroxyacetone to glycerol. In
`N certain aspects, the yeast cell is of the genera Kluyoeromyces, Candida or Issatchenkia. In other aspects, the yeast cell is capable
`of producing at least one organic acid, such as lactate. The yeast cells produce significantly less glycerol than the wild—type strains,
`and usually produce greater yields of desired fermentation products. Yeast cells of the invention often grow well when cultivated,
`despite their curtailed glycerol production.
`
`BUTAMAX 1028
`
`BUTAMAX 1028
`
`

`

`WO 2007/106524 A2
`
`|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`Published:
`— without international search report and to be republished
`upon receipt of that report
`
`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes and Abbreviations ” appearing at the begin—
`ning of each regular issue of the PCT Gazette.
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`YEAST CELLS HAVING DISRUPTED PATHWAY FROM
`DIHYDROXYACETONE PHOSPHATE TO GLYCEROL
`
`This invention was made under contract no. DE-FCS6-03G013145 with the
`
`United States Department of Energy. The United States Government has certain
`
`rights to this invention.
`
`This application claims priority from United States Provisional Application
`
`No. 60/781,674, filed 13 March 2006.
`
`10
`
`This invention relates to certain genetically modified yeast, and fermentation
`
`processes to produce lactic acid using. those genetically modified yeast.
`Yeast are used as biocatalysts in a number of industrial fermentations. There
`
`is an increasing interest in using yeast to ferment sugars to organic acids such as
`
`the
`As more organic acid is produced in these fermentations,
`lactic acid.
`fermentation medium becomes increasingly acidic. Most bacteria that produce these ‘
`
`.15
`
`organic acids do not perform well in strongly acidic environments—they either do not
`
`survive under those conditions or else produce so slowly that the process becomes -
`
`economically unviable. As a result, it becomes necessary to buffer the medium to
`
`maintain a higher pH. This causes difficulty in recovering the product in acid form.
`
`20
`
`It is preferred to conduct the fermentation at a lower pH at which the product is
`
`partially or wholly in the acid form.
`Yeast
`species have been considered as candidates
`
`for
`
`such low—pH
`
`fermentations. Many yeast speciesnaturally ferment hexose sugars to ethanol, but
`
`few if any naturally produce significant yields of organic acids such as lactic acid.
`
`25
`
`Accordingly, efforts have been made to genetically modify various yeast species to
`
`insert one or more genes that will enable the cell to produce lactic acid. In order to
`
`divert sugar metabolism from ethanol production to lactic acid production, these cells
`
`have also been genetically modified to disrupt or delete the native pyruvate
`
`decarboxylase (PDC) gene. This work is described, for example, in‘WO 99/14335, WO
`
`30
`
`00/71738 A1, WO 02/42471 A2, WO 03/049525 A2, WO 03/102152 A2 and WC)
`
`'
`03/102201 A2.
`Glycerol is produced in significant yield in many of these yeast fermentations.
`
`Glycerol may serve as an osmoprotectant for the cell. Glycerol formation may help
`
`regenerate redox cofactors under fermentation conditions.
`Glycerol is produced in many yeast cells by metabolizing dihydroxyacetone
`
`phosphate (DI-IAP).
`
`In most yeast species, DHAP is reduced by a glycerol-3-
`
`-1-
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`phosphate dehydrogenase (GPD, systematic name sn-glycerol-3-phosphate:NAD+ 2-
`
`oxidoreductase, EC 1.1.1.8) enzyme to form glycerol-S-phosphate (GBP). G3P-serves
`
`as a precursor ‘ for
`
`lipid biosynthesis as well as a glycerol precursor. G3? is
`
`dephosphorylated to glycerol by a glycerol-3-phosphatase enzyme (GPP, systematic
`
`name glycerol-l-phosphate phosphohydrolase, EC 3.1.3.21).
`
`There exists an alternate pathway for glycerol production, which is important
`
`for some yeast, such as S. pombe. In this pathway, dihydroxyacetone phosphate is
`
`phosphate
`dihydroxyacetone
`by
`dihydroxyacetone
`into
`dephosphorylated
`phosphatase. Dihydroxyacetone is then converted into glycerol in conjunction with
`NADH oxidation by NADH+-dependent glycerol dehydrogenase (systematic name
`
`glycerolzNAD+ 2-oxidoreductase, EC 1.1.1.6).
`
`Because glycerol production consumes carbon that could otherwise be used to
`
`produce a more desirable fermentation product, this glycerol' production represents a
`
`significant source of yield loss. In addition, glycerol production comes at the expense
`
`of both ATP and NADH. This directs energy away fiom the production of biomass or
`the desired product. For both .of these reasons, it would be desired to reduce or
`eliminate glycerol production by the cell. A further consideration is that the
`reduction or elimination of glycerol production could simplify recovery and
`
`10
`
`15
`
`purification of the desired product.
`
`20
`
`A Saccharomyces cerevisiae strain has been genetically engineered to delete its
`
`native GPD genes, thus depriving the cell of the GPD enzyme and preventing glycerol
`production.
`See Nissen et al., “Anaerobic and aerobic batch cultivations of
`Saccharomyces cerevisiae mutants impaired in glycerol synthesis”, Yeast, 2000:
`
`16:463-474. Nissen et al. report that the mutated cells grew very poorly under both
`
`25
`
`anaerobic and aerobic conditions when both of the native GPD genes were disrupted. ~
`
`According. to Nissen et al., the mutated cells produced much less glycerol than the
`
`wild-type cells. Nissen et al. hypothesized that the poor growth seen in the double
`
`deletant strains was due to a depletion of the cell’s NAD+ pool, because glycerol
`
`production was not available to oxidize NADI-I in the cell.
`
`30
`
`It would be desirable to provide a yeast cell that produces a desired organic
`
`product, which produces little or no glycerol, and which also grows well under aerobic
`conditions, anaerobic conditions or both aerobic and anaerobic conditions.
`
`In one aspect, this invention is a mutant yeast cell of a pre-whole genome
`
`duplication yeast species, having a deletion or disruption of a native metabolic
`
`-2-
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`pathway from dihydroxyacetone phosphate to glycerol. The deletion or disruption of
`the native metabolic pathway may include a deletion or disruption of at least one
`native glycerol-S-phosphate dehydrogenase (GPD) gene. The deletion or disruption of
`
`the native metabolic pathway'may include .a deletion or disruption of at least one
`
`native glycerol—B-phosphatase (GPP) gene. It may include a deletion or disruption of
`at least one native g1ycerol-3-phosphate dehydrogenase (GPD) gene and at least one
`
`native glycerol-B-phosphatase (GPP) gene. The deletion or disruption of the native
`
`metabolic pathway may include a deletion or disruption of at least one native
`dihydroxyacetone phosphate phosphatase gene, native glycerol dehydrogenase gene,
`
`10
`
`or both.
`
`In another aspect, this invention is a mutant yeast cell of of a pre-Whole
`
`genome duplication yeast species, which mutant cell produces less than 2.0 g/L of
`
`glycerol when cultivated under the following standard microaerobic conditions:
`
`A. defined aqueous medium containing, at the start of cultivation, 5 g/L ammonium
`sulfate, 3 g/L potassium dihydrogen phosphate, 0.5 g/L magnesium sulfate, trace
`
`15
`
`elements, vitamins, 150 g/L glucose;
`
`B. pH at the start of cultivation of 3.5, with fermentation medium being buffered if
`necessary to prevent the pH from falling below 3.0 or rising above 7.0 during the
`
`cultivation;
`
`20
`
`C. Cultivation inoculated with the yeast cell to an ODsoo of 1.0;
`
`D. Cultivation temperature 30°C;
`
`E. Cultivation continued until glucose concentration is reduced to 10 g/L, but is not
`continued. for more than 120 hours;
`F. Aeration and agitation sufficient to produce an oxygen uptake rate of 5.0 i- 1.0
`
`25
`
`mmol/L/hr-
`'
`In another aspect, this invention is a mutant yeast cell of a pre-whole genome
`duplication yeast species, which lacks the ability to produce an active glycerol-3-
`phosphate dehydrogenase (GDP) enzyme. For purposes of this invention, a cell is
`considered to lack the ability to produce an active enzyme if the activity of such
`
`30
`
`enzyme in the cell is reduced by at least 75%, preferably at least 90%, compared to
`
`the activity of that enzyme in the wild-type strain. Enzyme activity of any particular
`enzyme can be determined using appropriate assay methods. Commercial assay kits
`are available for determining g1ycerol-3-phOSphate dehydrogenase activity. An
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`example of such a product is designated as MK426 by Takara Bio, Inc. and is
`available through Fisher Scientific, Pittsburgh, Pennsylvania.
`
`In another aspect, this invention is a mutant yeast cell of a pre-whole genome
`
`duplication yeast species, which lacks the ability to produce an active glycerol-3-
`
`phosphatase enzyme.
`
`In another aspect, this invention is a mutant yeast cell which lacks the ability
`
`to produce an active dihydroxyacetone phosphate phosphatase enzyme that is
`
`natively produced by wild type cells of the yeast species, lacks the ability to produce
`
`an active NADH+-dependent glycerol dehydrOgenase enzyme that
`
`is natively
`
`10
`
`produced by wild type cells of the yeast species, or both
`
`In another aspect, this invention is. a mutant yeast cell that is genetically
`
`modified to produce a product organic acid, said yeast cell further having a deletion or
`
`disruption of a native metabolic pathway fiom dihydroxyacetone phosphate to
`
`glycerol and a deletion or disruption of a native metabolic pathway from pyruvate to
`
`15
`
`ethanol. The deletion or disruption of the native metabolic pathway may include a
`
`'deletion or disruption of at least one native glycerol—B-phosphate dehydrogenase gene:
`
`The deletion or disruption of the native metabolic pathway may include a deletion or
`
`disruption of at least one native glycerol-S-phosphatase gene.
`
`It may include a
`
`deletion or disruption of at least one native glycerol-3-phosphate dehydrogenase gene
`
`20
`
`and at least one native glycerol-B-phosphatase gene.
`
`Cells in accordance with the invention have been found to produce very low
`
`levels of glycerol when cultivated under fermentation conditions. Glycerol production
`has been found to be below 0.2 g/L under a range .of fermentation conditions.
`Surprisingly, the cells of the invention grow well under fermentation conditions,
`despite the lack of glycerol production and in some embodiments despite the lack of
`
`25
`
`glycerol-3-phosphate production. The cells of the invention have also been found to
`
`have improved acid tolerance in some instances. Accordingly, the invention is also a
`
`fermentation process wherein a cell of any of the foregoing aspects of the invention is
`cultivated under fermentation conditions to produce a fermentation product, wherein
`
`30
`
`the yield of carbon source to glycerol is less than 2% by weight.
`
`Figure 1 is a diagram depicting the pBH158 plasmid.
`Figure 2 is a diagram depicting the pBI-I159 plasmid.
`
`Figure 3 is ‘a diagram depicting the pBH16O plasmid.
`
`-4-
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`Figure 4 is a diagram depicting the pBHlGl plasmid.
`. Figure 5 is a diagram depicting the pMM28 plasmid.
`
`Figure 6 is a diagram depicting the pM1318 plasmid.
`
`Figure 7 is a diagram depicting the pM1321 plasmid.
`
`Figure 8 is a diagram depicting the pM1355 plasmid.
`
`Figure 9 is a diagram depicting the pMI357 plasmid.
`
`Figure 10 is a diagram depicting the pMI433 plasmid.
`
`Figure 11 is a diagram depicting the pMI449 plasmid.
`Figure 12 is a diagram depicting the pMI454 plasmid.
`
`10
`
`Figure 13 is a diagram depicting the pBI-I165 plasmid.
`
`Figure 14 is a diagram depicting the pTMCGl plasmid.
`
`The yeast cells of the invention are made by performing certain genetic
`
`modifications to a host yeast cell. The host yeast cell is one which, as a Wild-type
`
`15
`
`strain, is natively capable of metabolizing at least one sugar to glycerol. The native
`
`metabolic pathway may involve a metabolic pathway from dihydroxyacetone
`
`phosphate to glycerol-S-phosphate to glycerol. The native pathway may involve a
`
`metabolic pathway from dihydroxyacetone phosphate to dihydroxyacetone to glycerol.
`
`Host cells may contain both of those native metabolic pathways.
`
`20
`
`The term “native,” when used herein with respect to genetic materials (e.g., a '
`
`gene, promoter, terminator or other DNA sequence), refers to genetic materials that
`are found (apart from individual-to-individual mutations which do not affect function)
`
`within the genome of Wild-type cells of that species of yeast. “Native capability” (and
`
`its variations such as “natively capable”) indicates the ability of wild-type cells to
`
`25
`
`perform the indicated function. For example, a cell is natively capable of metabolizing
`
`‘ a sugar to glycerol if wild-type cells of that species possess that capability prior to any
`
`genetic modifications. A gene is considered to be “functional” Within a cell if it
`
`functions within the cell to produce an'active protein. A “native pathway” or “native
`
`metabolic pathway” refers to a metabolic pathway that exists and is active in wild-
`
`30
`
`type cells of that species of yeast. An enzyme is “natively produced” by a yeast
`species if the enzyme is produced in active form by wild type cells of that species of
`
`yeast.
`
`In this invention, “exogenous” means with respect to any genetic material that
`
`it is not native to the host cell.
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`Suitable host yeast cells for certain embodiments of the invention include
`yeast cells which are not descended from a line that underwent the ancient (~100
`million years ago) whole genome duplication event described 'by Wolf et al.,
`
`“Molecular evidence for an ancient duplication of the entire yeast genome”, Nature
`387, 708-713 (1997) (hereinafter “Wolf et al 1997”), Langkjaer et al., “Yeast genome
`duplication was followed by asynchronous differentiation of duplicated genes”, Nature
`
`421, 848-852 (2003) and Merico et al., “Fermentative lifestyle in yeasts belonging to
`
`the Saccharomyces complex”, FEBS Journal 274, 967-989 (2007) (hereinafter “Merico
`
`2007”). Such yeast cells are instead descended from one or more other lines of yeast
`
`10
`
`cells that existed at the time of the whole genome duplication event, and are referred
`to herein as “pre-Whole genome duplication yeast”. The whole genome duplication
`event
`is seen as critical
`for
`the evolution of the fermentative capabilities of
`
`Saccharomyces cerevisiae and other species descended from the common ancestor in
`
`which the genome duplication occurred (Merico 2007).
`
`Included in the set of genes
`
`15
`
`duplicated in the genome duplication are those encoding glycerol-B-phosphate
`
`dehydrogenase
`
`and g1ycerol-3-phosphatase,
`
`as are. genes
`
`encoding fumarate
`
`reductase which is also involved in maintaining redox balance (Wolfe et a1 1997).
`
`Among
`
`the
`
`suitable
`
`pre-whole
`
`genome
`
`duplication yeast
`
`cells
`
`are
`
`hemiascomycetous yeast cells. Hemiascomycetous yeast are single—celled yeast
`
`20
`
`classified within the order Saccharomycetales.
`
`Other suitable yeast cells include those falling Within any of the clades 7, 8, 9,
`
`10, 11, 12, 13 or 14 of the Saccharomyces complex, as described in Figure 9 (p. 430) of
`
`Kurtzman and Robnett,
`
`"Phylogenetic
`
`relationships
`
`among yeasts
`
`of
`
`the
`
`'Saccharomyces complex‘ determined from multigene sequence analyses", FEMS
`
`25
`
`Yeast Res. Vol. 4, pp. 417-432. (2003), incorporated herein by reference. Those clades
`
`are designated by the names Zygosaccharomyces, Zygotorulaspora, Torulaspora,
`Lachancea, Kluyveromyces, Eremotheciunt, Hanseniaspora and Saccharomycodes,
`respectively, in Merico 2007, supra, and in Kurtzmann, “Phylogenetic circumscription
`
`of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae .
`
`. .”
`
`30
`
`FEMS Yeast. Res. Vol. 4, pp. 233-245 (2003) (hereinafter “Kurtzman 2003”).
`
`Other suitable yeast cells include (but are not limited to) yeast cells classified
`
`under the genera Candida, Saccharomyces, Schizosaccharomyces, Kluyveromyces,
`
`Pichia, Issatchenkia, and Hansenula.
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`_A class of host cells that are of particular interest includes any of those of a
`species contained within the I. orientalis/I.
`terricola clade: Members of the I.
`orientalis /I. terricola clade are identified by analysis of the variable D1/D2 domain of‘
`
`the 26S ribosomal DNA of yeast species, using the method described by Kurtzman
`
`and Robnett in “Identification and Phylogeny of Ascomycetous Yeasts from Analysis
`
`of Nuclear Large Subunit (26S) Ribosomal DNA Partial Sequences”, Antonie van
`
`Leeuwenhoek 73:331-371, 1998,
`
`incorporated herein by reference (hereinafter
`
`“Kurtzman and Robnett 1998”).
`
`See especially p. 349 and 361. Analysis of the
`
`variable D1/D2 domain of the 268 ribosomal DNA from hundreds of ascomycetes has
`
`10
`
`revealed that the I. orientalis/I.
`
`terricola clade contains closely related species.
`
`Members of the I. orientalis/I.
`
`terricola clade exhibit greater similarity in the
`
`variable D1/D2 domain of the 268 ribosomal DNA to that of other members of the
`
`clade than to that of yeast species outside of the cladea. Therefore, other members of
`
`the I. orientalis/I.
`
`terricola clade can be identified by comparison of the D1/D2
`
`15
`
`domains of their respective ribosomal DNA and comparing to that of other members
`
`of the clade and.closely related species outside of the clade, using Kurtzman and
`
`Robnett’s methods. Yeast species within the I. orientalis/I. terricola clade are all
`
`hemiascomycetous yeast within the broader Pichia/Issatchenkia/Saturnispbra/
`Dekkera clade. Another class of host cells of interest is the Lorientalis/P. fermentans
`
`20
`
`clade as described by Kurtzman and Robnett 1998. .That clade is the most terminal
`clade that contains at least the species Issatchenkia orientalis, Pichia galeiformis,
`Pichia sp. YB-4149 (NRRL designation), Candida ethanolica, P. deserticola, P.
`
`membranifaciens and P. fermentans.
`
`Other host cells of particular interest are any of those of a species contained
`within the Kluyveromyces clade of Saccharomyces complex, as described (as Clade 11)
`
`25
`
`in Figure 9 (p. 430) of Kurtzman and Robnett, "Phylogenetic relationships among
`
`yeasts of
`
`the ‘Saccharomyces complex' determined from multigene sequence
`
`analyses". FEMS Yeast Res. Vol. 4, pp. 417-432. (2003), incorporated herein by
`
`reference, and in Figure
`
`1 of Kurtzmann, “Phylogenetic circumscription of
`
`30
`
`Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae .
`
`.
`
`.”
`
`FEMS Yeast. Res., Vol. 4,
`
`.pp. 233-245 (2003)
`
`(hereinafter “Kurtzman 2003”),
`
`incorporated herein-'by reference. The Kluyveromyces clade includes at least the
`
`species S. kluyveri, K. aestuaryii, K. nonfermentans, K. lactic, K. marxianus and K.
`
`

`

`WO 2007/106524
`
`PCT/US2007/006408
`
`dobzhanskii, and would include additional species classifiable within that clade using
`the multigene sequene analysis methods described in Kurtzman 2003.
`
`Such yeast cells are of particular interest when genetically modified to
`produce an organic acid,
`especially lactate. Host cells
`from the Candida,
`
`Kluyveromyces and Ittatchenkia genera are generally preferred. Host cells from the
`
`,Kluyveromyces
`
`and I. orientalis/P.
`
`fermentans
`
`clades described before
`
`are
`
`particularly preferred, in those embodiments where the mutant cell produces an
`
`organic acid, as well as in cases where the mutant cell produces another fermentation
`
`product (such‘as, for example, ethanol) in addition to or instead of an organic acid.
`
`10
`
`Especially preferred host cells are C. sonorensis, K. marxianus, K. thermotolerans, C.
`
`methanosorbosa, and I. orientalis. Most preferred cells are K. marxianus, C.
`
`sonorensis, and I. orientalis. When first characterized, the species I. orientalis was
`
`assigned thetname Pichia kudriavzevii. The anamorph (asexual form) of I. orientalis
`
`is known as Candida krusei. Suitable strains of K. marxianus and C. sonorensis
`
`15
`
`include those described in W0 00/7 17 38 A1, WO 02/42471 A2, WO 03/049525 A2, W0
`03/102152 A2 and W0 03/102201A2. Suitable strains of I. orientalis are ATCC strain
`
`‘32196 and ATCC strain PTA-6648.
`
`By “deletion or disruption” of a metabolic pathway, it means that the pathway
`
`is either rendered completely inoperative, or else its activity is reduced by at least
`
`20
`
`75%, preferably at least 90%, relative to the wild-type cell. Activity of a pathway may
`
`be reduced by reducing the amount of active enzyme that is produced, by reducing the
`
`activity of the enzyme that is produced, or some combination of both. By “deletion or
`
`disruption” of a gene it is meant that the entire coding region of the gene is
`
`eliminated (deletion), or the coding region of the gene, its promoter, and/or its
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`25
`
`terminator region is modified (such as by deletion, insertion, or mutation) so that the
`
`gene no longer produces an active enzyme, the gene produces a severely reduced
`
`quantity (at least 75% reduction, preferably at least 90% reduction) of the active
`
`enzyme, or the gene produces an enzyme with severely reduced (at least 75% reduced,
`
`preferably at least 90% reduced) activity.
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`30
`
`In most cases, the deletion or disruption of the native metabolic pathway will
`involve a deletion or disruption of at least one GPD gene, at least one GPP gene, or
`
`both.
`
`In cells such as S. pombe, that have an alternate metabolic pathway based on
`
`dihydroxyacetone phosphate phosphatase and glycerol dehydrogenase, the deletion or
`
`disruption of the native metabolic pathway will usually include a deletion or
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`-8-
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`WO 2007/106524
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`PCT/US2007/006408
`
`glycerol
`gene,
`dihydroxyacetone phosphate phosphatase
`the
`disruption of
`dehydrogenase .gene, or both. In cells having both pathways, deletions or disruptions
`of both pathways can be performed.
`
`The term “glycerol—3—phosphate dehydrogenase gene” and “GPD gene" are
`
`used herein to refer to (a) any gene that encodes for a protein with glycerol-3-
`
`phosphate dehydrogenase activity and/or (b) any chromosomal DNA sequence that
`
`encodes for an enzyme that is at least 50%, preferably at least 60% and more
`
`preferably at least 65% identical to any of the amino acid sequences identified as
`
`SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5,
`
`10
`
`SEQ. ID. NO. 6, or SEQ. ID. NO. 7. “Glycerol—3—phosphate dehydrogenase activity”
`
`refers to the ability of a protein to catalyze the reaction of DI-IAP to glycerol-3-
`
`phosphate. For purposes of this invention, percent identity of amino acid sequences
`
`of DNA, RNA or proteins can conveniently computed using BLAST (NCBI Basic Local
`
`Alignment Search Tool) version 2.2.1 software with default parameters. Sequences
`
`15
`
`having an identities score of at least XX%, using the BLAST version 2.2.13 algorithm
`
`with default parameters, are considered at least XX% identical. The BLAST software
`
`is available from the National Center for Biological Information, Bethesda, Maryland.
`
`Similarly, “glycerol-3-phosphatase gene” and “GPP gene” are used herein to
`
`designate (a) any gene that encodes for a protein with glycerol-B-phosphatase activity
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`20
`
`and/or (b) any chromosomal DNA sequence that encodes for a protein that is at least
`
`'50%, preferably at least 60% and more preferably at least 65% identical to any of the
`
`amino acid sequences identified as SEQ. ID. No.8, SEQ. ID. NO. 9, SEQ. ID. NO. 10,
`
`SEQ. ID. NO 11 or SEQ- ID. NO 12. “G1ycerol-3-phosphatase activity” refers to the
`
`ability of a protein to catalyze the dephosphorylation of glycerol—S—phosphate to form
`
`25
`
`30
`
`glycerol.
`The term “dihydroxyacetone phosphate phosphatase” gene is used herein to
`
`denote any gene that encodes for a protein with dihydroxyacetone phosphate
`
`phosphatase activity. “Glycerol dehydrogenase” gene is used herein to denote (a) any
`gene coding for a protein with glycerol dehydrogenase/ activity and/or (b) any
`chromosomal DNA sequence that encodes for a protein that
`is at least 50%,
`
`preferably at least 60% and more preferably at least 65% identical to the amino acid
`
`sequence identified as SEQ. ID. NO. 13. “Dihydroxyacetone phosphate phosphatase
`
`activity” refers to the ability of a protein to catalyze the reaction of dihydroxyacetone
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`

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`WO 2007/106524
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`PCT/US2007/006408
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`“Glycerol dehydrogenase activity” refers to the
`phosphate to dihydroxyacetone.
`ability of a protein to catalyze the reduction of dihydroxyacetone to glycerol.
`‘The deletion or disruption of any of the foregoing genes can be accomplished
`
`by forced evolution, mutagenesis, or genetic engineering methods,
`
`followed by
`
`appropriate selection or screening to identify the desired mutants.
`
`In mutagenesis methods cells are exposed to ultraviolet radiation or a
`mutagenic substance, under conditions sufficient to achieve a high kill rate (60—
`
`99.9%, preferably 90-99.9%) of the cells. Surviving cells are then plated and selected
`
`or screened for cells having the deleted or disrupted metabolic activity. Cells having
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`10
`
`the desired mutation can be screened for on the basis of their reduced ability to
`
`produce. glycerol. Disruption or deletion of any of the foregoing genes can be'
`
`confirmed through PCR or Southern analysis methods.
`
`Genetic engineering to delete or disrupt the metabolic pathway to glycerol is
`
`conveniently accomplished in one or more steps via the design and construction of
`
`15
`
`appropriate deletion constructs and transformation of the host cell with those
`
`constructs. The term “construct” is used herein to denote a DNA sequence that is
`
`used to transform a cell. The construct may be, for example, in the form of a circular
`
`plasmid or vector, in the form of a linearized plasmid or vector, may be a portion of a
`circular plasmid or vector (such as is obtained by digesting the plasmid or vector with
`one or more restriction enzymes), or may be a PCR product prepared using a plasmid
`
`2O
`
`or vector as a template.
`
`Selection or screening follows to identify successful
`
`transformants. Electroporation and/or chemical (such as calcium chloride— or lithium
`
`acetate—based) transformation methods can be used.
`
`..
`
`The following discussion of deletion constructs is equally applicable to the
`
`25
`
`deletion or disruption of any of the g1ycerol-3—phosphate dehydrogenase, glycerol-8-
`
`phosphatase, dihydroxyacetone phosphate phosphatase or glycerol dehydrogenase
`
`genes.
`
`A deletion construct is conveniently assembled by first cloning two DNA
`sequences of the target gene and/or its upstream (5’) or downstream (3’) flanking
`
`30
`
`regions. The sequences are preferably non-contiguous, but may be contiguous if
`additional genetic material (such as a selection marker cassette) is to be interposed
`
`between them on the construct. In this context, “non-contiguous” means that the
`DNA sequences are not immediately adjacent to each other in the Wild-type genome,
`
`but instead are separated from each other in the wild-type genome by an area that is
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`WO 2007/106524
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`to be deleted in order to delete or disrupt the gene.
`“Contiguous” sequences are
`directly adjacent to 'each other in the Wild-type genome. One‘of the sequences may
`include a region 5’ to the promoter of the target gene, all or a portion of the promoter
`region, all or a portion of target gene coding region, or some combination thereof. The
`other sequence may include a region 3’ to the terminator of the target gene, all or a
`
`portion of the terminator region, and/or all or a portion of the target gene coding
`
`region. .A deletion construct is then produced containing the two sequences oriented
`
`in the same direction in relation to each other as they natively appear on the
`
`10
`
`chromosome of the host cell. Typically a selection marker is cloned between the
`sequences to allow selection of transformants; as described more fully below. This
`construct is used to transform the host cell. Electroporation and/or chemical (such as
`
`calcium chloride- or lithium acetate-based) transformation methods can be used.
`
`In successful transformants, a homologous recombination event at the locus of
`
`the target gene results in the disruption or the deletion of the functional gene. All or
`
`15
`
`a portion of the native target gene, its promoter and/or terminator is deleted during
`
`this recombination event. If the deletion construct contains genetic material between
`the two sequences taken from the target locus (such as a selection marker cassette or
`
`structural gene cassette), that genetic material is inserted into the host cell’s genome
`
`at the locus of the deleted material. Analysis by PCR or Southern analysis can be
`
`20
`
`performed to confirni that the desired deletion has taken place.
`
`It is usually desirable that the deletion construct may also include a functional
`
`selection marker cassette. When a single deletion construct is used, the marker
`
`cassette resides on the vector downstream (i.e., in the 3’ direction) of the 5’ sequence
`
`from the target locus and upstream (i.e., in the 5’ direction) of the 3’ sequence from
`
`25
`
`the target locus. Successful transformants will contain the selection marker cassette,
`
`which imparts to the successfully transformed cell some characteristic that provides a
`
`basis for selection. A “selection marker gene” is one that encodes a protein needed for
`
`the survival and/or growth of the transformed cell in a selective culture medium.
`Typical selection marker genes encode proteins that
`(a) confer
`resistance to
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`30
`
`antibiotics or other
`
`toxins,
`
`(such as,
`
`for example,
`
`zeocin (Streptoalloteichus
`
`hindustanus ble bleomycin resistance gene), G418 (kanamycin—resistance gene of
`
`Tn903) or hygromycin (aminoglycoside antibiotic resistance gene from E. coli)), (b)
`
`complement auxotro