WO2004085627A1 - Nouvelles souches de saccharomyces cerevisiae qui utilise du xylose - Google Patents

Nouvelles souches de saccharomyces cerevisiae qui utilise du xylose Download PDF

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WO2004085627A1
WO2004085627A1 PCT/SE2004/000404 SE2004000404W WO2004085627A1 WO 2004085627 A1 WO2004085627 A1 WO 2004085627A1 SE 2004000404 W SE2004000404 W SE 2004000404W WO 2004085627 A1 WO2004085627 A1 WO 2004085627A1
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xylose
anaerobic
growth
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saccharomyces cerevisiae
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Marco Sonderegger
Uwe Erich Sauer
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Forskarpatent I Syd Ab
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present invention relates to novel Saccharomyces cerevisiae strains utilizing xylose for production of ethanol, as well as method for selecting a xylose-utilizing Saccharomyces cerevisiae strain.
  • metabolic engineering became a standard for strain improvement and has been very successful when simple cellular traits were targeted (Ostergaard et al . 2000; Stafford and Stephanopoulos 2001).
  • genomics age with the associated genome-wide analytical technologies gives further impetus to rational approaches, metabolic engineering of more complex or not fully understood cellular systems remains a challenge.
  • combinatorial approaches such as directed evolution in contemporary protein engineering evolutionary approaches are becoming increasingly important to augment metabolic engineering of complex phenotypes. In certain cases, however, even seemingly simple metabolic systems resist straightforward rational engineering.
  • An additional puzzling fact is the inability of xylose to support anaerobic growth in both natural and recombinant xylose-utilizing yeasts. Since many bacteria can grow anaerobically on xylose, the reason for this inability is not really understood at present, but has been ascribed to a general restriction of eucaryotic xylose metabolism to respirative conditions.
  • the present invention relates to novel xylose-utilizing Saccharomyces cerevisiae strains, in particular to those deposited at DSMZ under the accession numbers DSM 15519 and DSM 15520 on the 18th of March, 2003.
  • DSM 15519 relates to the strain coded TMB3001C1 below
  • DSM 15510 relates to the strain coded TMB3001C5 below.
  • the invention relates to new xylose-utilizing Saccharomyces cerevisiae mutant strain overexpressing the xylose-utilizing pathway by overexpressing xylose reductase (XR), xylitol dehydrogenase (XDH), and xylokinase (XK), wherein one or more of genes encoding NADPH producing enzymes are up-regulated and one or more of the genes encoding NADH consuming enzymes are up-regulated.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XK xylokinase
  • the genes GAL2, PYK2, PUT4, GPD2, and ADH4 are over- expressed.
  • regulators of glycolysis and central carbon metabolism GCR1, RAP1, REB1, PDC2, HAP1, HAP5, and SNF1 are over-expressed.
  • GAL3 a regulator of galactose metabolism: GAL3 is over- expressed.
  • regulators of osmotic stress response (HOG-pathway) : SSK22, RIM15, ASK10, SKN7, PBS2, MSN4, MSN1, and PTP2 are over-expressed.
  • regulators with other functions: TEA1, YAP5, ACAl, CLN3, RIOl, and SNF2 are over-expressed.
  • genes YATl, YCR060W, YEL057C, YIL176C, MCH5, and ATM1 are expressed to improve the capability of anaerobic growth on xylose.
  • the genes encoding for xylose reductase (XR), and xylitol dehydrogenase (XDH) are over expressed to a higher degree than in the corresponding non-mutated strain TMB3001.
  • Another aspect of the invention relates to a method for selecting an improved xylose- utilizing Saccharomyces cerevisiae strain, which strain overexpresses the xylose-utilizring pathway by overexpressing xylose reductase (XR), xylitol dehydrogenase (XDH), and xylokinase (XK), which method is characterized in that a starting Saccharomyces cerevisiae strain is cultured under aerobic conditions in minimal medium containing xylose as sole carbon source, is selected with regard to growth on xylose, is continued cultured under microaerobic conditions in minimal medium containing xylose as sole carbon source, is selected with regard to growth on xylose, and is finally cultured under anaerobic conditions in minimal medium containing xylose as sole carbon source to produce a xylose-utilising Saccharomyces cerevisiae strain.
  • XR xylose reductase
  • XDH
  • random mutagenesis using ethyl methane sulfonate is carried out on the starting Saccharomyces cerevisiae strain during the aerobic culture phase.
  • random mutagenesis using ethyl methane sulfonate is carried out on the Saccharomyces cerevisiae strain selected after the aerobic culture phase.
  • selection is done using continuous culture.
  • selection is done using chemostat selection.
  • culturing in a continuous culture takes place at constant dilution rate.
  • the dilution rate is 0.05 h "1 .
  • the first selection is made after at least 50 generations culturing in minimal medium using xylose as only carbon source, the second selection is made after further at least 100 generations, and the third selection is made after further at least 120 generations.
  • the culturing under aerobic conditions takes place under at least 90 generations, preferably 130 generations.
  • culturing under microaerobic conditions takes place under further at least 110 generations, preferably at least 140 generations. In one preferred embodiment, culturing under anaerobic conditions takes place under further at least 150 generations, preferably 190 generations.
  • single clones from anaerobic growth on xylose plates are selected, and plated on anaerobic minimal medium containing xylose as the sole carbon source, and plates are incubated in sealed environment, to provide for an anaerobic atmosphere.
  • a pre-selection step is carried out prior to the aerobic culture on sole xylose medium comprising an aerobic growth on a minimal medium containing glucose and xylose as carbon sources in a molar ratio of about 50: 50.
  • yeast cultures were grown at 30°C in minimal medium containing per litre: 5 g (NH 4 ) 2 S0 4 , 3 g KH 2 P0 4 , 0.5 g MgS0 4 »7H 2 0, 15 mg EDTA, 4.5 mg ZnS0 4 »7H 2 0, 0.3 mg CoCI 2 »6H 2 0, 1 mg MnCI 2 «4H 2 0, 0.3 mg
  • the medium was supplemented with ergosterol (Fluka) and Tween 80 (Sigma) for anaerobic cultivation. Both components were dissolved in boiling 99.8% (v/v) ethanol and were added to the medium at a final concentration of 0.01 g I "1 and 0.42 g I "1 , respectively. Solid media were prepared by adding 1.5% (w/v) technical Agar (Becton Dickinson).
  • Standard molecular biology techniques were used to clone the phosphoketolase (xfp), the phosphotransacetylase (pta) and the acetaldehyde dehydrogenase (Ehadh2) genes under the strong truncated HXT7 promotor in the yeast multicopy plasmids p426HXT7, p424HXT7 and p425HXT7, respectively.
  • the Bifidobacterium lactis xfp gene was sub-cloned by ligation of the 2.6 kbp DNA fragment, resulting from the EcoRI-Hindlll digestion of the pFPK ⁇ plasmid, after gel extraction (QUIAEX II, QUIAGEN, Basel, Switzerland).
  • the Entamoeba histolytica Ehadh2 containing 3 kbp cDNA fragment was sub-cloned from the BamHI-Xbal digestion of pET3a-ehadh2 into the BamHI-Spel digested p425HXT7 plasmid.
  • the Bacillus subtiiis pta gene was amplified with taq DNA polymerase (Promega, Madison, WI) by PCR (Cycle: lx 2 min. at 95°C; 30x (lmin at 95°C, 0.5 min at 58°C, 1.25 min at 74°C); 5 min at 74°C) from B.
  • Plasmids were transformed in S. cerevisiae by the lithium acetate method using the S.c. EasyComp transformation kit (Invitrogen, Carlsbad, CA).
  • Chemostat selections were performed in a Sixfors 6-minireactors system (Infors, Botmingen, Switzerland) at a dilution rate (D) of 0.05 h "1 and mixing at 300 rpm.
  • D dilution rate
  • a constant working volume of 300 ml was maintained by continuously removing excess culture broth through a needle that was fixed at a predetermined height.
  • the culture pH was maintained at 5.0 ⁇ 0.3 by supplementing the minimal medium with 50 mM potassium hydrogen phthalate (Fluka). Aerobic conditions were installed by aeration at a rate of 0.3 I min "1 . Microaerobic conditions were installed by stepwise reduction of aeration until no measurable flow was seen in the reactor effluent gas.
  • Microaerobic conditions were defined as ⁇ lml/min per 300 ml vessel containing medium and biomass, i.e. a severely oxygen depleted condition. Anaerobic conditions were established by slight sparging ( ⁇ 1 ml min "1 ) with technical N 2 ( ⁇ 200 ppm 0 2 ; independently quantified with a Prima 600 mass spectrometer (Fisons Instruments, Uxbridge, England)). It should be noted that due to the contaminating 0 2 , these conditions are not strictly anaerobic. To ensure robust long-term operation of up to 4 months, marprene tubing (Ismatech, Glattbrugg, Switzerland) was used with external peristaltic pumps for feeding and harvesting. Contamination controls were done in two weeks intervals by plating culture aliquots on YPD medium (10 g I "1 yeast extract, 20 g I "1 peptone, and 20 g I "1 glucose) plates and by microscopic analysis.
  • YPD medium 10 g I "1 yeast
  • Hungate tubes which are 17 ml Pyrex glass tubes that are sealed with butyl rubber septa and plastic screw caps (Bellco Glass Inc., Vineland, NJ). Cultures were grown in minimal medium, containing 10 g I "1 xylose as the sole carbon source. New cultures were inoculated when the growth rate declined, which occurred typically after about 1 week.
  • Aerobic cultures were grown in 500 ml baffled shake flasks with 50 ml minimal medium at 300 rpm on a rotary shaker and 30°C.
  • TMB3001 To adapt TMB3001 to aerobic growth on sole xylose, it was first grown on YPX medium ( 10 g I "1 yeast extract, 20 g I "1 peptone, and 20 g I '1 D- xylose), then on YNB xylose medium (6.7 g I "1 Yeast Nitrogen Base and 20 g I "1 D-xylose), and finally once in minimal medium with sole xylose prior to inoculation.
  • YPX medium 10 g I "1 yeast extract, 20 g I "1 peptone, and 20 g I '1 D- xylose
  • YNB xylose medium 6.7 g I "1 Yeast Nitrogen Base and 20 g I "1 D-xylose
  • Fermentation performance was evaluated in anaerobic batch cultures containing 50 g I "1 each of glucose and xylose. The concentrations of all other minimal medium components except KH 2 P0 4 were doubled. To avoid major drops in pH, 100 mM citric acid buffer (pH 5.5) was added, which maintained the pH above 4.7 in all cases. Cultures were grown in 175 ml serum bottles, filled with 150 ml medium and stirred magnetically at 100 rpm and 30°C. Anaerobic (but not strictly anaerobic) conditions were maintained by slight continuous sparging (1-2 bubbles sec "1 ) with technical N 2 (0 2 ⁇ 200 ppm) (PanGas, Dagmersellen, Switzerland).
  • Inocula were prepared by growing frozen stock cultures first on YPD medium and finally in minimal medium with 20 g I "1 glucose. Strictly anaerobic growth experiments on sole xylose were done in Hungate tubes or serum bottles sealed with butyl-rubber septa by sparging the basic salt solution of the minimal medium with pure N 2 (0 2 ⁇ 5 ppm) (PanGas) for 15 min. After autoclaving, the remaining filter-sterilized, N 2 -sparged medium components and 10 g I "1 xylose were added.
  • Stocks for strain maintenance were generated from overnight cultures grown in YPD medium by adding glycerol to a final concentration of 15% (w/v) and were then stored at - 80°C. To preserve the original clonal composition of selection chemostats, population aliquots from the selection cultures were frozen directly, without intermediate batch growth.
  • EMS ethyl metha ne sulfonate
  • RNA extraction was performed by the hot-phenol-method, quantified and checked for high quality at 260 and 280 nm with a spectrophotometer. Formaldehyde containing agarose gels were used to assess RNA integrity. mRNA isolation, cDNA synthesis, in vitro transcription, cRNA fragmentation, hybridization (GeneChip YG-S98 Arrays, Affymetrix, Santa Clara, CA), array washing, staining and scanning were performed by the SWEGENE Micro-array Resource Centre (Lund, Sweden). Data acquisition, processing and comparison analysis was performed with The Microarray Suite Software, version 5.0 (Affymetrix) and the
  • Cell growth was monitored by following the optical density at 600 nm (OD 600 ) or by determining the Klett-value with a Klettmeter (Bel-Art Products, Pequonock, NJ).
  • Cellular dry weight (cdw) was determined from 10 ml culture aliquots that were centrifuged at 5,000 rpm for 20 min in pre-weighed glass tubes, washed once with water, and dried at 110°C for 24 h to constant weight.
  • Commercially available kits were used for enzymatic determination of glucose (Beckman), xylose (Medichem, Steinenbronn, Germany), xylitol (R-Biopharm, Darmstadt, Germany), acetate (R-Biopharm), and glycerol (Sigma).
  • Ethanol concentrations were determined by gas chromatography (5890E chromatograph; Hewlett- Packard) with a Permabond-CW20M-0.25 column (Macherey-Nagel) and butyrate as the internal standard. C0 2 and ethanol concentrations in the reactor offgas were determined with a Prima 600 mass spectrometer (Fisons Instruments, Uxbridge, England).
  • the specific xylose uptake rate was determined as the ratio of the linear regression coefficient of xylose concentration versus time and the average biomass concentration between the onset of xylose consumption and about 100 h after inoculation. In these cases, the OD 6 oo-to-cdw correlation was determined at the end of each fermentation.
  • biomass and by-product yields were determined as the ratio of the produced molar carbon amount for a considered product and the molar amount of total carbon sources consumed during steady state. A ratio of 0.476 gC gBiomass "1 was assumed. Specific consumption and production rates were calculated as the ratio of the considered molar production rates and the steady state biomass concentration. The amount of evaporated ethanol was measured and considered. To determine intracellular metabolic fluxes, a modified preexistent stoichiometric model was used.
  • TMB3001C1 exhibited significantly slower aerobic glucose catabolism with a 36% reduced growth rate and a 48% reduced specific rate of glucose uptake, when compared to TMB3001 (Table 1). Although the efficiency of exponential growth on glucose was not affected, as judged from the maximum bioma ss yield (Y x s), the maximum biomass concentration attained by TMB3001C1 was significantly lower.
  • TMB3001C1 During aerobic growth on sole xylose, both clones grew significantly faster than their parent, but TMB3001C1 grew by far the most rapid (Table 2). Surprisingly, only TMB3001C1 but not TMB3001C5 was capable of strict anaerobic growth on sole xylose in Hungate tubes (Fig. 5). Further increased strength of anaerobiosis by addition of cysteine (Table 3) or Na 2 S (data not shown) had no significant impact on the growth of TMB3001C1. Compared to the evolved population, which also includes this clones, TMB3001C1 grew significantly faster on xylose under anaerobic conditions.
  • TMB3001 0.44 a 3.14 ⁇ 0.05 0.14 b 2.1 c TMB3001C5 0.41 2.61 ⁇ 0.09 0.16 2.2 TMB3001C1 0.28 1.62 ⁇ 0.02 0.17 1.4
  • SD Standard deviation
  • TMB3001C5 0.064 0.13 ⁇ 0.00 0.50 ⁇ 0.02 1.9 TMB3001C1 0.119 0.27 ⁇ 0.02 0.45 ⁇ 0.04 2.0
  • TMB3001C1, TMB3001C5 and TMB3001 were cultivated the strains TMB3001C1, TMB3001C5 and TMB3001 in chemostat cultures at D 0.05 h "1 for transcriptome analysis (Table 4).
  • TMB3001C1 and TMB3001 were analyzed on 10 g I '1 glucose and 10 g I '1 xylose under both aerobic and anaerobic conditions, whereas TMB3001C5 showed no significant differences to TMB3001 under these conditions (data not shown), and was therefore not considered.
  • TMB3001C1 and TMB3001 were analyzed on 10 g I '1 glucose and 10 g I '1 xylose under both aerobic and anaerobic conditions, whereas TMB3001C5 showed no significant differences to TMB3001 under these conditions (data not shown), and was therefore not considered.
  • TMB3001C1 and TMB3001C5 were investigated under the latter conditions.
  • TMB3001 TMB3001C1 (Cl)
  • TMB3001C5 C5
  • D 0.05 h '1 Physiological parameters of TMB3001 (TMB), TMB3001C1 (Cl) and TMB3001C5 (C5) during chemostat cultivations (D 0.05 h '1 ) for transcriptome analysis.
  • Xylose cone. 1 (g/L) 4.287 0.277 0.826 0.725 7.381 4.723 q(Glucose) b (gGlc/(gX.h)) 0.070 0.059 n.d. n.d. 0.492 0.568 q(Xylose) b (gxyl/(gx.h)) 0.042 0.061 0.114 0.127 0.140 0.313 q(EtOH) a (gEtOH/(gX.h)) 0.002 0.001 0.001 n.d. 0.244 0.287
  • TMB3001C1 depleted completely the fed xylose in steady state under aerobic conditions in both presence and absence of glucose as co-substrate. Under these conditions, increased xylose consumption was correlated with growth, whereas under anaerobic conditions a doubled specific xylose uptake rate, without xylose depletion in steady state, could be observed. In the latter case, increased xylose catabohc rate was correlated with doubled glycerol yield and decreased biomass formation. As TMB3001C1, TMB3001C5 was abl e to consume completely xylose in the aerobic steady state on this sole carbon source.
  • pyruvate kinase 2 glycerol-3-phosphate dehydrogenase and alcohol dehydrogenase 4 indicates that the mutants try to improve intracellular metabolic fluxes in central carbon metabolism by increasing expression levels of limiting enzymes, and by lowering intracellular NADH levels, what increases xylitol dehydrogenase activity.
  • Different regulatory proteins are listed in the first cluster.
  • the second cluster contains also the hxt2 high affinity hexose transporter.
  • TMB3001C1 shows tendentially increased expression levels of NADPH producing and NADH consuming enzymes.
  • the galactose permease seems to be the most relevant xylose transporter which is strongly overexpressed, whereas significantly changed expression profiles of different regulators were observed (data not shown). Correlations between regulators and genes with changed expression profiles remain to be established.
  • the first yeast strain that grows on xylose as the sole carbon source under strict anaerobic conditions is described above.
  • Such strains were isolated from a long-term, multi-step chemostat evolution experiment, which was initiated with the metabolically engineered S. cerevisiae strain TMB3001 that overexpresses the xylose-utilization pathway of P. stipitis (Eliasson et al. 2000).
  • the selection procedure was based on the well-known evolution of mutants with increased substrate affinity and utilization in chemostat cultures.
  • the key to successful evolution was to decouple selection for aerobic and anaerobic xylose utilization (Fig. 1).
  • the selective pressure was adjusted to the present capabilities of the evolving culture, allowing advantageous mutations to accumulate under growth permissive conditions.
  • the clones described here were isolated after 460 generations or 266 days of selection, the ability to grow anaerobically on sole xylose was first detected after 270 generations, immediately after switching the culture conditions to anaerobiosis ( ⁇ 200 ppm 0 2 ).
  • the achieved phenotype of the best xylose-utilizing clone TMB3001C1 with a maximum specific growth rate of 0.012 h "1 and a biomass yield of 0.021 g g "1 during strict anaerobic growth on xylose, represents by no means a final stage of evolution.
  • the anaerobic growth rate of TMB3001C1 could be more than doubled within 40 generations of batch culture selection (Fig. 6).
  • the rate of anaerobic xylose metabolism is still relatively slow, the isolation of these improved clones argues against the view that eucaryotic xylose metabolism is necessarily tied to respiration.
  • Our results are more consistent with the view that anaerobic growth on xylose does not naturally occur in yeasts because the rate of xylose metabolism is too slow, so that the rate of ATP production is insufficient (K ⁇ tter and Ciriacy 1993; Hahn-Hagerdal et al. 2001).
  • the population consisted of at least two subpopulations with distinct phenotypes, thus evidencing population heterogeneity (or polymorphism) that is often observed during evolution experiments.
  • the best representative of these, TMB3001C5 exhibited a 60% higher specific xylose uptake rate and a four-fold higher aerobic growth rate on sole xylose, when compared to TMB3001 (Table 2).
  • TMB3001C1 The best representative of this subpopulation, TMB3001C1, exhibited a more than three-fold higher specific xylose uptake rate and an eight-fold higher aerobic growth rate on xylose, when compared to TMB3001 (Table 2). All class II clones grew slower and less efficiently on glucose than TMB3001 and exhibited significantly increased overflow metabolism to acetate and glycerol (Fig. 4), indicating a drastic reorganization of central metabolism.
  • the class I clone TMB3001C5 can grow on ethanol, and possibly other metabolic by-products of the class II clones, with the contaminating 0 2 as an external electron acceptor.
  • the class II clone TMB3001C1 grows significantly faster then the D of the anaerobic selection chemostat when cultivated under conditions with contaminating 0 2 .
  • This view is also consistent with the obvious absence of a strong selection pressure for high anaerobic growth rate on xylose during chemostat selection, since faster growing clones were readily selected within comparatively few generations in strict anaerobic batch culture.
  • the applied strategy is a fruitful combination of rational metabolic engineering to render a strain amenable for selection and evolutionary techniques.
  • two industrial ethanol- producing strains were metabolically engineered with the same xylose-utilization pathway that was used here (Zaldivar et al. 2002).
  • the evolved strains shown here accumulate less xylitol and some clones have higher xylose consumption rates (e.g. TMB3001C1).
  • the engineered industrial strains produced only about 8% more ethanol than TMB3001 from a mixture of glucose and xylose (Zaldivar et al. 2002), while our best clone TMB3001C5 produced about 19% more ethanol than TMB3001 (Fig. 3).
  • the presented evolutionary engineering of enabling or improving substrate utilization is not confined to the recombinant strain used here, but can in principle be applied to other substrates or organisms; e. g. the above industrial strains.
  • the organism subjected to selection should have the genetic potential to utilize the new substrate. Evolution may then be used to improve substrate utilization or to improve it under novel conditions. While simpler traits may be directly selected for, more complex, multi-gene modifications require an evolution approach for step-wise improvements (Sauer 2001).
  • a particular aspect of the invention relates to the exploration of TMB3001C1 (DSM 15519). Strains, media, and cultivation conditions
  • the S. cerevisiae strain TMB3001 (CEN.PK 113-7A (MATa, his3- ⁇ l, MAL2-8C, SUC2) /7/ ' s3;;YIpXR/XDH/XK) (Eliasson et al., 2000) and its evolved mutant Cl (Sonderegger and Sauer, 2003) were used throughout. Cultures were stored in aliquots supplemented with 15% glycerol at -80°C and were revived by growth in YPD medium (10 g I "1 yeast extract, 20 g I "1 peptone, and 20 g I "1 glucose).
  • Shake flask cultures were grown in 500 ml baffled shake flasks with 50 ml medium at 30°C and 300 rpm. Carbon-limited chemostat cultures were grown in 1 I medium in a 2 I stirred tank reactor (Bioengineering, Wald, Switzerland) at a dilution (growth) rate of 0.05 h "1 . The volume was kept constant by continuous removal of excess culture broth through a sterile needle that was fixed at a predetermined height. A constant pH of 5.0 was maintained by automatic addition of 2 M KOH. Sparging with air at a rate of 1 I min "1 established aerobic conditions, whereas sparging with pure N 2 (0 2 ⁇ 5 ppm) at a rate of 0.35 I min "1 established anaerobiosis.
  • Constant gas flow rates were controlled by a mass flowmeter (Inceltech, Toulouse, France). To ensure anaerobiosis, the feed medium was also maintained under a N 2 atmosphere. The stirrer speed was set to 1,000 or 500 rpm under aerobic or anaerobic conditions, respectively. Culture aliquots for metabolic flux and transcript analysis were withdrawn in physiological steady state, defined as stable cell density and rate of C0 2 evolution for at least three volume changes.
  • mRNA isolation, cDNA synthesis, in vitro transcription (cRNA synthesis), and cRNA fragmentation were performed according to the Affymetrix expression analysis technical manual.
  • Hybridization, washing, staining, and scanning of the Gene Chip Yeast Genome S98 Arrays were done in a hybridization oven (Affymetrix), the Fluidics Station 400 (Affymetrix), and the GeneArray Scanner (Affymetrix).
  • the fold-change indicates the relative change in transcript levels when compared to a reference culture of TMB3001, and was used to identify genes that were differentially expressed in the two mutants. Differential gene expression analysis was done with the Significance Analysis of Micro-arrays (SAM) EXCEL add-in software (Tusher er al., 2001). For this purpose, fold-changes were considered statistically significant above a factor of two at a false positive rate of 1% (Piper et al., 2002).
  • DW Cellular dry weight
  • Ethanol, acetoin, and butanediol concentrations were determined by GC as described before (Sauer er al. , 1996). Pyruvate and succinate concentrations were determined by HPLC (Perkin Elmer, Shelton, Connecticut), with a Supelco H column (Supelco, Bellefonte, PA) and 0.15% H 3 P0 as the mobile phase. C0 2 and ethanol concentrations in the reactor off-gas were determined with a Prima 600 mass spectrometer (Fisons Instruments, Uxbridge, England).
  • V x S Ethanol, xylitol, acetate, glycerol, and butanediol yields were calculated by linear regression of by-product concentration versus substrate concentration, their specific production rates were calculated as the product of specific xylose or glucose uptake rate and the by-product yield.
  • biomass and by-product yields were determined as the ratio of the molar carbon in the considered product and the total molar carbon in the consumed substrates in steady state, assuming a ratio of 0.476 g(C) g(biomass) "1 (Wahlbom er al. , 2001).
  • Specific consumption and production rates were calculated as the ratio of the considered molar production rates and the steady state biomass concentration.
  • the fraction of evaporated ethanol, 0 2 , and C0 2 in the bioreactor off-gas were determined by on-line MS analysis.
  • the macromolecular cell composition was assumed to be 39% (w/w) polysaccharides, 50% (w/w) protein, and 6% (w/w) RNA in chemostat culture, and 40% (w/w) polysaccharides, 52% (w/w) protein, and 3% (w/w) RNA as well as 31% (w/w) polysaccharides, 56% (w/w) protein, and 9% (w/w) RNA in batch cultures on xylose and glucose, respectively (Wahlbom er al., 2001).
  • Cell extracts were prepared from mid-exponential growth phase cultures in minimal medium with glucose. Cell pellets were harvested by centrifugation, washed with deionized water, and resuspended in a 0.1 M triethanolamine buffer (pH 7.0), containing 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithio-threitol, and 0.5 mM EDTA. The suspension was vortexed with glass beads (0.5 mm diameter) at 4°C for 5 min, incubated on ice for 5 min, and vortexed again for 5 min. Cell debris and glass beads were separated by centrifugation at 20,000 x g and 4°C for 5 min.
  • Aerobic glucose/xylose TMB3001 0.16 glucose/xylose Cl 0.13 xylose Cl 0.34
  • GAL7 YBR018 226 248 242 Galactose-1-P uridyl transferase
  • HEM YDR232 2 2.1 1 5-aminolevulinate synthase
  • This Cl mutant provides a unique opportunity to elucidate the molecular mechanisms that are required for eukaryotic xylose metabolism under anaerobic conditions.
  • DNA microarray Conway and Schoolnik, 2003
  • metabolic flux analysis Sauer et al., 1996; Varma and Palsson, 1994; Wahlbom et al., 2001
  • balancing of redox equivalents is one crucial component, we provide strong evidence that it is ultimately the rate of ATP formation that limits anaerobic growth on xylose.
  • RNA microarray analysis was done with RNA isolated from the above chemostat cultures to elucidate molecular changes that underlie the capability to grow on xylose as the sole carbon source.
  • transcript levels were quantified from duplicate experiments with average coefficients of variation between 0.13 and 0.34 (Table 9).
  • 577 genes exhibited greater or equal than two-fold differential expression pattern (Piper et al., 2002) in Cl when compared to TMB3001 under at least one cultivation condition, 119 of which were differentially expressed under all three conditions (supplementary material).
  • differential gene expression was determined by comparison with TMB3001 under the same condition but with the additional carbon source glucose, since TMB3001 cannot grow at this rate on xylose (Hamacher et al. , 2002; Sonderegger and Sauer, 2003).
  • HXT5 and HXT2 were down-regulated under all conditions and HXT4 and STL1 under aerobic conditions (supplementary material).
  • HXT16 gene in contrast, was strongly up- regulated (Table 10).
  • HOG high osmolarity glycerol
  • MTH1 negative regulator of hexose transport
  • strains overexpressing the preferentially NADPH-dependent xylose reductase from P. stipitis generated the required NADPH primarily through the oxidative pentose phosphate pathway (Jeppsson et al. , 2002).
  • This causes lower glycolytic fluxes and the concomitantly reduced NADH re-oxidation by reactions downstream of fructose-6-P can be compensated in respiring but not in fermenting yeast (Bakker et al. , 2001); hence explaining the capacity of such recombinant strains to grow aerobically but not anaerobically on sole xylose (Eliasson et al. , 2000; Ho et al., 1998).
  • Adding the NADH-oxidizing compound acetoin to Cl cultures increased the anaerobic growth rate on sole xylose by about one third, which demonstrates that balancing of redox co-factors was still a growth-limiting problem for Cl. While the rate of xylose uptake remained constant, xylose catabolism increased because less of the side-product xylitol was produced, which demonstrates clearly that xylose uptake was not limiting. Flux analysis revealed that the specific cytosolic NADPH production rate remained constant but that the ATP production rate increased by the same factor as the growth rate in the acetoin co-feed culture.
  • the Cl mutant has apparently evolved such that altered redox cofactor metabolism in a number of reactions and higher expression levels of almost all catabolic genes permits higher catabolic fluxes of xylose to ethanol, which in turn provides ATP at a sufficient rate for growth under anaerobic conditions.
  • Transcriptome and intracellular metabolic flux analysis show clearly that, mainly under anaerobic conditions, the improved xylose catabolism observed in the mutant able to grow anaerobically on sole xylose increases absolute intracellular metabolic fluxes in the entire central carbon metabolism. This effect is correlated with higher expression levels of the involved enzymes, but also by a generalized increased expression of the majority of the NADPH producing and NADH consuming enzymes. This latter effect can be interpreted as an attempt of the mutant cell to increase flux through xylose reductase and xylitol dehydrogenase catalyzed reactions, lowering the negative effect caused by the redox imbalance generated by the different cofactor specificity of the two enzymes.
  • FIG. 1 Evolution of S. cere visiae TMB3001 in carbon-limited chemostat cultures at D of 0.05 h "1 under aerobic conditions with 5 g I "1 xylose and 1 g I "1 glucose (A); aerobic, microaerobic (light gray background), and anaerobic (dark gray background) conditions with 5 g I "1 xylose (B); and anaerobic conditions with 5 g I "1 xylose (C).
  • Airflow reduction from 0.3 I min "1 to ⁇ 1 ml min "1 (2) shut-off of airflow, and (3) onset of anaerobiosis by sparging with technical N 2 .
  • the evolving population was subjected to EMS mutagenesis prior to inoculation of the chemostats A, B, and C.
  • FIG. 2 Fermentation profile of TMB3001 (A), the 460-generation selection (B), clone TMB3001C5 representing the first phenotypic class (C), and clone TMB3001C1 representing the second phenotypic class (D), during anaerobic growth on 50 g I "1 glucose and 50 g I "1 xylose. Glucose and xylose consumption phases are highlighted by I and II, respectively. Gray shading indicates simultaneous consumption of glucose and xylose.
  • FIG. 3 Physiological parameters during anaerobic growth on 50 g I "1 glucose and 50 g I "1 xylose of TMB3001, the 460-generation population, and 15 clones isolated from this population. Maximum growth rate and biomass yield were determined during exponential growth on glucose (A). Specific xylose uptake rate and xylitol yield on xylose were determined between glucose depletion and 100 h of fermentation (B). The final ethanol concentration was determined at 180 h (C). Values for TMB3001 and the population are average values from duplicate experiments. Hairlines indicate the reference values of TMB3001.
  • FIG. 4 Yields of acetate (A) and glycerol (B) on glucose (black bars) and xylose (open bars) during anaerobic growth on 50 g I "1 glucose and 50 g I "1 xylose of TMB3001 and selected clones from both phenotypic classes. Yields on glucose were determined between inoculation and the begin of xylose uptake. Yields on xylose were determined between glucose depletion and 130 h. Values were determined from single experiments.
  • FIG. 5 OD 60 o and xylose concentration during strict anaerobic growth of TMB3001C1 in minimal medium with xylose as sole carbon source.
  • FIG. 6 Strict anaerobic growth rates on xylose minimal medium of 20 clones that were isolated after seven serial anaerobic batch cultures on xylose.
  • the hairline indicates the growth rate of the parental TMB3001C1 before selection.
  • FIG.7 Comparison of absolute intracellular metabolic fluxes (ovals, mMol gDW “1 h “1 , S.D. ⁇ 10%) and transcript levels (Boxes, arbitrary unit) of TMB3001 (upper values) and TMB3001C1 (lower values) during anaerobic chemostat cultivation on 10 g I “1 glucose and 10 g I "1 xylose. Only genes with significant expression changes are shown.
  • Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Appl. Environ. Microbiol. 68 : 1604-1609.
  • Yeast 8 501-517. Walfridsson M, Hallborn J, Penttila M, KerDenen S, Hahn-Hagerdal B (1995) Xylose- metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TALI genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase.

Abstract

L'invention concerne une souche mutante de saccharomyces cerevisiae qui utilise du xylose. Cette souche surexprime la réductase de xylose, la déshydrogénase de xylitol et la xylokinase. Elle concerne, en outre, un ou plusieurs gènes codant des enzymes produisant du NADPH et un ou plusieurs gènes codant des enzymes consumant du NADH surrégulés, ainsi qu'un procédé destiné à sélectionner une souche améliorée de S. cerevisiae, cultivée avec du xylose comme unique source de carbone dans des conditions aérobies, puis dans des conditions microaérobies et anaérobies. Chaque condition de culture s'étend sur plusieurs générations et ,est suivie d'une sélection de croissance sur le xylose. Enfin, une mutagenèse aléatoire est mise en oeuvre durant ou après la phase aérobie.
PCT/SE2004/000404 2003-03-26 2004-03-19 Nouvelles souches de saccharomyces cerevisiae qui utilise du xylose WO2004085627A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2005091733A2 (fr) * 2004-03-26 2005-10-06 Forskarpatent I Syd Ab Traits de souches de saccharomyces cerevisiae a croissance de xylose recombinees par analyse de transcription genomique
US7598374B2 (en) 2004-03-26 2009-10-06 Purdue Research Foundation Processes for the production of xylitol
WO2011003893A1 (fr) 2009-07-10 2011-01-13 Dsm Ip Assets B.V. Production par fermentation d'éthanol à partir de glucose, de galactose et d'arabinose employant une souche de levure recombinée
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WO2012049179A2 (fr) 2010-10-13 2012-04-19 Dsm Ip Assets B.V. Polypeptides à activité kinase
WO2012067571A1 (fr) * 2010-11-15 2012-05-24 Scandinavian Technology Group Ab Nouvelles souches de saccharomyces cerevisiae
US20130224815A1 (en) * 2010-11-11 2013-08-29 Toyota Jidosha Kabushiki Kaisha Method for producing ethanol using recombinant yeast strain
WO2013148993A1 (fr) 2012-03-30 2013-10-03 Novozymes North America, Inc. Procédés de fabrication de produits de fermentation
US20130273555A1 (en) * 2010-05-05 2013-10-17 Mascoma Corporation Detoxification of Biomass Derived Acetate Via Metabolic Conversion to Ethanol, Acetone, Isopropanol, or Ethyl Acetate
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WO2015148272A1 (fr) * 2014-03-28 2015-10-01 Danisco Us Inc. Voie de cellule hôte modifiée pour la production améliorée d'éthanol
JP5796138B2 (ja) * 2012-10-10 2015-10-21 Ｊｘ日鉱日石エネルギー株式会社 キシロースからエタノールを生産する酵母
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WO2016012429A1 (fr) 2014-07-24 2016-01-28 Dsm Ip Assets B.V. Cellule de levure présentant un transport amélioré du pentose
WO2016062875A2 (fr) 2014-10-23 2016-04-28 Novozymes A/S Variants de glucoamylase et polynucléotides les encodant
WO2016087327A1 (fr) 2014-12-01 2016-06-09 Novozymes A/S Polypeptides ayant une activité pullulanase comprenant les domaines x25, x45 et cbm41
US9528117B2 (en) 2009-07-24 2016-12-27 Dsm Ip Assets B.V. Fermentative glycerol-free ethanol production
WO2018015304A1 (fr) 2016-07-21 2018-01-25 Novozymes A/S Variants de sérine protéase et polynucléotides codant pour ceux-ci
WO2018015303A1 (fr) 2016-07-21 2018-01-25 Novozymes A/S Variants de sérine protéase et polynucléotides les codant
WO2018102348A1 (fr) 2016-11-29 2018-06-07 Novozymes A/S Variants d'alpha-amylase et polynucléotides codant pour ces derniers
EP3415624A1 (fr) 2014-01-22 2018-12-19 Novozymes A/S Variants de pullulanase et polynucleotides les codant
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US10435721B2 (en) 2016-12-21 2019-10-08 Creatus Biosciences Inc. Xylitol producing metschnikowia species
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WO2021089877A1 (fr) 2019-11-08 2021-05-14 Dsm Ip Assets B.V. Procédé de production d'éthanol
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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ELIASSON A. ET AL.: "Anaerobic Xylose Fermetation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 66, no. 8, August 2000 (2000-08-01), pages 3381 - 3386, XP000973771 *
HAHN-HAGERDAL B. ET AL.: "Metabolic Engineering of Saccharomyces cerevisiae for Xylose Utilization", ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY, vol. 73, 2001, pages 53 - 84, XP002980022 *
HO N.W.Y. ET AL.: "Genetically Engineered Saccharomyces Yeast Capable of Effective Cofermentation of Glucose and Xylose", APPLIED AND ENVIRONMENT MICROBIOLOGY, vol. 64, no. 5, May 1998 (1998-05-01), pages 1852 - 1859, XP002980023 *
WAHLBOM C.F. ET AL.: "Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054", FEMS YEAST RESEARCH, vol. 3, 2003, pages 319 - 326, XP002980002 *
WAHLBOM C.F. ET AL.: "Molecular Analysis of a Saccharomyces cerevisiae Mutant with Improved Ability To Utilize Xylose Shows Enhanced Expression of Proteins Involved in Transport, Initial Xylose Metabolism, and the Pentose Phosphate Pathway", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 69, no. 2, February 2003 (2003-02-01), pages 740 - 746, XP002966584 *
WALFRIDSSON M. ET AL.: "Xylose-Metabolizing Saccharomyces cerevisiae Strains Overexpressing the TKL1 and TAL1 Genes Encoding the Pentose Phosphate Pathway Enzymes Transketolase and Transaldolase", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, no. 12, December 1995 (1995-12-01), pages 4184 - 4190, XP002053306 *

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