EP0973882A1 - OBTENTION D'$i(ESCHERICHIA) COLI RESISTANTES A L'ETHANOL HAUTEMENT CONCENTRE - Google Patents

OBTENTION D'$i(ESCHERICHIA) COLI RESISTANTES A L'ETHANOL HAUTEMENT CONCENTRE

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Publication number
EP0973882A1
EP0973882A1 EP98914390A EP98914390A EP0973882A1 EP 0973882 A1 EP0973882 A1 EP 0973882A1 EP 98914390 A EP98914390 A EP 98914390A EP 98914390 A EP98914390 A EP 98914390A EP 0973882 A1 EP0973882 A1 EP 0973882A1
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EP
European Patent Office
Prior art keywords
ethanol
microorganism
ethanologenic
microorganisms
liquid medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98914390A
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German (de)
English (en)
Inventor
Lonnie O. Ingram
Lorraine P. Yomano
Sean W. York
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Florida
University of Florida Research Foundation Inc
Original Assignee
University of Florida
University of Florida Research Foundation Inc
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Publication date
Application filed by University of Florida, University of Florida Research Foundation Inc filed Critical University of Florida
Publication of EP0973882A1 publication Critical patent/EP0973882A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Escherichia coli KOll is much less ethanol tolerant.
  • bacteria have been developed that have the ability to convert the sugars from, for example, lignocellulose to ethanol, the problem remains that et ⁇ anol tolerance m these bacteria limits boch the rate of ethanol production and the final ethanol concentration which can be achieved m the fermentors .
  • This invention is based upon the discovery that novel mutants of Escherichia coli KOll exhibit the ability co grow and survive m etnanol concentrations beyond that: in which the parent Escherichia coli KOll can survive.
  • the invention is also based upon the discovery of an improved ethanol selection process which alternates between selection for ethanol resistance m liquid medium and selection for rapid growth on solid medium containing a high level of chloramphenicol . This selection process resulted m novel strains of Escherichia coli KOll, as discussed above, which are useful for ethanol production.
  • mutants producing 20% more ethanol and completing fermentation more rapidly than the parental E. coli KOll strain, could be produced. Moreover, the mutants are capable of growth m up to 50 g/L ethanol while the parent is incapable of growth at 35 g/L ethanol. Finally, the mutants show dramatically enhanced survival exposure to 100 g/L ethanol as compared to the parent. These characteristics of the mutants means that the expense of ethanol production from lignocellulosic hydrolysates will decrease by achieving higher ethanol concentrations m shorter times and reducing the costs of nutrients, capital equipment, product recovery and waste disposal.
  • the invention comprises a method for the selection of ethanologenic microorganisms comprising contacting the microorganisms sequentially to a liquid medium and a solid medium, wherein said liquid medium is used to select for ethanol tolerance and said solid medium is used to select for ethanologenic microorganisms having the ability to grow and produce ethanol .
  • the invention comprises liquid media containing increasing concentrations of ethanol and solid media containing antibiotics and a fermentable sugar for use in the above selection process .
  • the invention comprises an ethanologenic microorganism having the ability to grow in ethanol concentrations of greater than 35 g/L.
  • the ethanologenic microorganism is selected from the group consisting of Erwinia, Klebsiella, Xanthomonas, Zymomonas and Escherichia, specifically K. oxytoca and E. coli .
  • the E. coli bacterium is selected from the group comprising LY01, LY02 and LY03.
  • the invention comprises an ethanologenic mutant having the ability to produce at least 10% more ethanol than the parental bacteria, preferably Escherichia coli KOll, under equivalent fermentation conditions.
  • Figure 1A shows the ability of mutants to grow in increasing concentrations of ethanol relative to the parental Escherichia coli KOll.
  • Figure IB shows the ability of mutants to survive in 10% ethanol (w/w) relative to the parental Escherichia coli KOll.
  • Figure 2A shows the ability of mutants, relative to the parental Escherichia coli KOll, to grow in the presence of glucose and no ethanol .
  • Figure 2B shows the ability of the LYOl mutants to grow in varying concentrations of ethanol and constant glucose relative to the parental Escherichia coli KOll strain in 3.5% ethanol and constant glucose.
  • Figure 2C shows the ability of mutants, relative to the parental Escherichia coli KOll, to grow in the presence of xylose and no ethanol .
  • Figure 2D shows the ability of the LYOl mutants to grow in varying concentrations of ethanol and constant xylose relative to the parental Escherichia coli KOll strain in 3.5% ethanol and constant xylose.
  • Figure 3A shows the osmotic tolerance of mutants to increasing concentrations of glucose relative to the parental Escherichia coli KOll.
  • Figure 3B shows the osmotic tolerance of mutants to increasing concentrations of xylose relative to the parental Escherichia coli KOll.
  • Figure 4A shows the ability of mutants to convert 14% glucose to ethanol relative to the parental Escherichia coli KOll.
  • Figure 4B shows the ability of mutants to convert 14% xylose to ethanol relative to the parental Escheri chia coli KOll.
  • Figure 5 shows a map of pLOI1531 and subclones . The map is of insert DNA from KOll. The vector is not shown .
  • Figure 6 is a map of pL0I1534 and subclones.
  • the invention relates to an ethanologenic mutant having improved ethanol tolerance.
  • the mutant can produce at least 10% more ethanol than the parental bacteria, (e.g. Escherichia coli KOll) when grown under equivalent conditions.
  • the mutant can grow in ethanol concentrations which exceed those of the parental microorganism.
  • a microorganism e.g., an ethanologenic microorganism
  • mutant of the subject invention can be produced by the process of (1) contacting the parental microorganism (e.g., an ethanologenic microorganism) with a first liquid medium comprising an aqueous solution comprising ethanol, selecting one or more microorganisms that survive; (2) contacting one or more microorganisms obtained from the preceding step with a solid growth medium for a sufficient period of time to permit growth.
  • This process can be repeated, such as two, three, four or more times to further improve ethanol tolerance. With each repeating step, the concentration of ethanol is incrementally increased.
  • the microorganism (s) obtained from step (2) can be contacted (step (3)) with a second liquid medium comprising an aqueous solution comprising an amount of ethanol greater than present in said first liquid medium, selecting one or more microorganisms that survive; and (4) contacting one or more microorganisms obtained from the preceding step with a solid growth medium for a sufficient period of time to permit growth.
  • the microorganisms selected in such a manner have improved ethanol tolerance to the parental microorganisms .
  • the liquid and solid mediums can contain additional components, as necessary or desirable.
  • the solid medium can include nutrients, such as sources of carbon, sulfur and nitrogen suitable for growth of the parental microorganism.
  • suitable growth medium include Luria broth and Basal Salts Media.
  • the solid medium will contain a sugar, such as xylose and/or glucose. This is desirable to ensure that the microorganisms selected form this step can produce ethanol from the sugar source in good to excellent yields (such as possessing the same or better ethanol production ability as the parent microorganism) .
  • the medium can also include an antibiotic, e.g. chloramphenicol , tetracycline, or ampicillin and a fermentable sugar.
  • the liquid media include an aqueous solution of ethanol.
  • the media can, optionally, contain nutrients as well, including a suitable carbon, sulfur and nitrogen source.
  • the liquid medium can also contain a buffer to control the pH of the medium.
  • the liquid media preferably contain sugar, such as xylose and/or glucose.
  • the first liquid medium, second liquid medium or both generally contain at least about 3.5% (by weight) ethanol.
  • the second and subsequent liquid media contain incrementally greater concentrations of ethanol.
  • the second liquid medium can contain at least about 4% (by weight) ethanol.
  • a third liquid medium (when present) can contain at least about 4.5% (by weight) ethanol.
  • the microorganisms which can be subjected to the above process can be prokaryotic or eukaryotic and include bacteria, yeasts and fungi.
  • the process is particularly suited for ethanologenic microorganisms (such as ethanologenic bacterium) which comprise one or more enzymes which convert a sugar (such as a pentose (e.g., xylose) or a hexose (e.g., glucose)) to ethanol.
  • suitable ethanologenic microorganisms comprise one or more nucleic acid molecules which encode alcohol dehydrogenase and pyruvate decarboxylase .
  • Ethanologenic bacteria comprising one or more nucleic acid molecules which encode alcohol dehydrogenase and pyruvate decarboxylase (as isolated from, for example, Zymomonas species, such as Zymomonas mobilis) are known. Also microorganisms that possess xylulokinase, transaldolase, transketolase and xylose isomerase are known (such as those expressed by enteric bacteria, such as Escherichia coli ) . Many microorganisms have the ability to convert both xylose and glucose to ethanol. Such organisms possess both sets of enzymes.
  • Ipet refers to the integration of Z. mobilis pdc and adhB genes into the chromosome.
  • pet refers to the presence of mobilis pdc and adhB genes in plasmid pLOI555.
  • pet refers to the presence of mobilis pdc and adhB genes in the indicated plasmid.
  • Cm r is the an E. coli shuttle vector carrying the cat gene
  • Ethanologenic microorganisms or host cells which can be employed for the insertion of enzymes which convert one or more sugars to ethanol, can be selected from bacteria, yeasts, fungi, or other cells. Suitable bacteria include Erwinia, Klebsiella, Xanthomonas,
  • Zymomonas such as Zymomonas mobilis
  • Escherichia Preferred species include K. oxytoca and E. coli .
  • gram-positive bacteria such as members of the genera Bacillus, for example, B . pumilus , B . subtilis and B . coagulans , members of the genera Clostridium, for example, Cl . acetobutylicum, Cl . aerotolerans, Cl . thermocellum, Cl . thermohydrosulfuricum and Cl . thermosaccharolyticum, members of the genera Cellulomanas like C. uda and Butyrivibrio fibrisolvens .
  • Acceptable yeasts for example, are of the species of Cryptococcus like Cr. albidus, Monilia, and Pichia stipi tis and Pullularia pullulans .
  • microorganisms can be subjected to the selection process of the claimed invention as they occur in nature or after isolation or genetic manipulation, as in mutation or genetic engineering.
  • soil or fecal samples containing microorganisms can be subjected to the described process.
  • the ethanologenic properties of the mutated microorganism can be introduced or further improved by inserting one or more enzymes which convert a sugar to ethanol, as described in the above patents.
  • an isolated ethanologenic microorganism with good to excellent ethanol producing properties are subjected to the above process.
  • the temperature and pressure of the process are generally not critical but should be selected to ensure viability and growth of the microorganisms. Suitable temperatures can be between about 20° and about 60°C. Pressure will generally be atmospheric.
  • the pH should also be selected towards the ability of the microorganism to remain viable and grow. For example, the pH may generally be selected to be between about 4.5 and 8.0.
  • the tolerance of a microorganism (or members of the species) can be determined readily and frequently are related to the conditions of the microorganism's native environment. Guidance for selecting optimal conditions for growth can be obtained for example in Bergey ' s Manual of Bacteriology.
  • the retention time of each step of the process is also not generally critical.
  • the time in which the microorganisms are subjected to the ethanol -containing liquid medium is generally selected such that some, but not all, of the population has died.
  • the step can last between about 8 hours to two weeks . Frequently, several days can be sufficient. Where the step lasts for several days, it may be desirable to exchange or add fresh liquid medium with the same or greater concentration of ethanol to the microorganisms.
  • the time for permitting growth of the microorganism on the solid medium is not generally critical and is dependent upon the microorganism. Slow growing microorganisms will require longer retention times than fast growing microorganisms, as generally known in the art. The time is generally long enough to differentiate colonies which are growing better than other colonies. Frequently, several days are sufficient for a fast growing bacteria.
  • the mutants produced by the claimed process have greater ethanol tolerance than the parental microorganisms.
  • the microorganisms can have improved ability to produce ethanol, as well.
  • the genetic basis for the mutation can be identified. For example, the chromosome and/or the RNA transcripts produced by the mutant microorganism can be compared to the parent microorganism. This can be done through hybridization technology, as described generally in Sambrook, et al . and Ausubel, et al . Once the genetic basis of the improved mutant has been identified, the mutation can be repeated or an equivalent produced through genetic engineering.
  • a recombinantly produced equivalent can be made by deleting the gene, deleting the regulatory sequences of the gene or targeting a site-specific mutation to shift the reading frame or remove an active site of the gene, as described in Ausubel, et al . and Sambrook, et al . In any event, the result is the inability of the microorganism to express an active gene product.
  • recombinantly produced equivalent can be made by substituting the native promoter with a stronger promoter of the gene, adding an enhancer or introducing more copies of the gene.
  • a recombinantly produced equivalent can be prepared by introducing the mutated sequence under the control of a promoter region recognized by the host cell.
  • mutants which do not express active cyclic AMP receptor protein, active biosynthetic alanine racemase or both have increased ethanol tolerance.
  • the invention includes microorganisms of increased ethanol tolerance wherein the microorganism does not express active cyclic AMP receptor protein, active biosynthetic alanine racemase or both. This can be achieved, for example, by such molecular biology techniques as site-specific mutagenesis and knocking out the gene, also known as "knock outs.” This can be accomplished, for example, by homologous recombination, described in Ausubel, et al . and Sambrook, et al .
  • Microorganisms which do not express active cyclic AMP receptor protein and/or active biosynthetic alanine racemase can be particularly suitable host cells for expressing enzymes (e.g., recombinantly) which convert a sugar to ethanol.
  • Suitable microorganisms for use in the invention are as discussed above.
  • Preferred microorganisms are bacteria, particularly gram-negative bacteria (e.g., enteric bacteria) such as Escherichia coli , Erwinia chrysanthia and Klebsiella oxytoca .
  • Particularly preferred microorganisms include those described in the U.S. Patents and applications to Ingram, et al . and Picataggio, et al . , above.
  • the invention further relates to methods of using the ethanol tolerant microorganisms described herein.
  • Microorganisms which have increased ethanol tolerance can be ethanologenic or not ethanologenic.
  • Ethanologenic microorganisms can be used in methods of producing ethanol, employing processes generally known in the art. Examples of suitable ethanol -producing processes include those described in the above patents and application to Ingram, et al . and Picataggio, et al . , which have been incorporated by reference.
  • An improved process for producing ethanol is described in copending application U.S.S.N. 08/833,435, by Ingram, et al . , filed April 7, 1997, which is also incorporated herein by reference.
  • ethanologenic microorganisms include the fermentation of sugar containing materials to foods and beverages.
  • ethanologenic microorganisms are employed in the manufacture of soy sauce, sake, beer and wine.
  • this invention can be employed to further improve the activities and ethanol tolerance of the microorganisms employed in these processes.
  • Microorganisms of the invention which are not ethanologenic or slightly ethanologenic are particularly useful as host cells for inserting nucleic acid molecules which encode one or more enzymes which catalyze one or more reactions in the glycolytic pathway, or other pathway which converts sugar to ethanol. That is, such microorganisms are particularly useful in the production of ethanologenic microorganisms through recombinant DNA technology.
  • E. coli KOll was used in these studies. This is an ethanol producing derivative of E . coli B in which the Zymomonas mobilis genes for ethanol production (pdc, adhB) have been integrated into the chromosome immediately upstream from chloramphenicol acyl transferase (ca t) (U.S. Patent No. 5,424,202). In this strain, resistance to chloramphenicol (600 mg liter "1 ) was used to select for high level expression of pdc and adhB . Cultures were grown in modified Luria broth containing per liter: 5 g NaCl, 5 g Yeast Extract, 10 g Tryptone, 40 or 600 mg chloramphenicol, and 20-140 g of fermentable carbohydrate.
  • KOll cultures were transferred daily by diluting 1:20 to 1:200 into 10 ml of fresh broth containing ethanol and glucose (50 g liter "1 ) in 18x150 mm culture tubes. Tubes were incubated for 24 h at 35°C without agitation. As cultures increased in density during subsequent transfers, ethanol concentrations were progressively increased to select for resistant mutants. Twice weekly, cultures were diluted and spread on solid medium to enrich for ethanol-resistant mutants which grew rapidly and retained high level expression of the Z . mobili s genes. Colonies on these plates were scraped into fresh broth and diluted. The dilutions were then used as inocula in ethanol -containing broth. KOll was initially transferred into 3.5% ethanol.
  • the ethanol concentration in the broth was increased to 4.0%; after 13 days, the ethanol concentration was increased to 4.5%; after 14 days, the ethanol concentration was increased to 5.0%. Dilutions into higher concentrations of ethanol did not appear to yield mutants with further increases in ethanol resistance which were also capable of rapid growth.
  • KOll. Colonies were transferred to 3 ml of broth and the resulting suspension diluted 60-fold into 13x100 culture tubes containing 0%, 4.5%, and 5% ethanol. After incubation for 24 h at 35°C, cell growth was measured as O.D. 550nm .
  • Mutants were maintained on plates containing 1% isopropanol and stored at -75°C in 40% glycerol .
  • Inocula were grown for 16 h (Beall, D.S. et al., "Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli . " Biotechnol . Bioeng. 38:296-303 (1991)) (30°C) without agitation in Luria broth containing glucose or xylose (50 g/L) . Cells were harvested by centrifugation (6000 x g, 5 min, ambient temperature) and added to initiate fermentation to provide 1.0 OD at 550 nm (approximately 330 mg liter "1 , dry cell weight) .
  • Genomic Library Chromosomal DNA was isolated from KOll essentially as described by Cutting, S.M. and Vander Horn, P.B., Genetic analysis, p. 37-74. In C.R. Harwood and S.M. Cutting (ed.), Molecular biological methods for Bacillus . John Wiley & sons Ltd., Chichester, England (1990) .
  • a genomic library of the parental strain, KOll was constructed by ligating Sau3AI partial digestion products (4-9 kbp fragments) into the BamHI site of pUC18 followed by transformation into DHS . The resulting library consisted of approximately 8,000 clones. After pooling these colonies, plasmids were isolated to produce a library. Standard procedures were used for the construction, isolation, transformation, and analysis of plasmids (Sambrook et al . , 1989) .
  • D-cycloserine was used to selectively kill cells capable of growing in 3.5% ethanol (w/w) while allowing non-growing cells to survive. At this concentration of ethanol, mutants continue to grow while the parent remains viable without increasing in cell number.
  • LY02 and LY03 were transformed with the KOll plasmid library and allowed to grow overnight into colonies. Recombinant colonies of each mutant were harvested by scraping into Luria broth containing glucose (50 g liter "1 ) and 3.5% ethanol, inoculated to provide 0.1 O.D. at 550 nm, and incubated at 35°C. After 1.5 hr, D-cycloserine (100 mg liter "1 ) was added and the incubation continued for 4 hours. After 4 h, CFU/ml had dropped by over 95%. Clones harboring putative genes for decreased ethanol tolerance were isolated from these plates .
  • the QIAprep spin plasmid kit (Qiagen, Chatsworth, CA) was used for plasmid purification. Dideoxy sequencing was performed using fluorescent primers [forward, 5 ' CACGACGTTGTAAAACGAC-3 ' (SEQ ID NO:l); reverse, 5 ' -ATAACAATTTCACACAGGA-3 ' (SEQ ID NO : 2 ) ] (LI -COR, Lincoln, NE) .
  • Extension reactions were performed with a Perkin Elmer GeneAmp PCR System 9600 (Norwalk, CT) using an Excel Sequencing Kit-LC (Epicentre Technologies, Madison, WI) (30 cycles; denaturation for 30 sec at 95°C, annealing for 30 sec at 60°C, and extension for 1 min at 70°C) . Extension products were separated and read with a LI -COR DNA Sequencer model 4000L.
  • EXAMPLE 1 Isolation of Ethanol-tolerant Mutants of E. Coli KOll Using the materials and procedures outlined above, a total of 135 colonies were initially tested for growth in 4.5% (w/w) ethanol, a concentration at which the parental KOll failed to grow. Forty three colonies were turbid after 24 h incubation at 35°C and were saved for further testing.
  • FIG. 1A As shown in Figure 1A, after 24 h, the mutants were much more resistant to ethanol than the parent KOll.
  • Figure 2B, Figure 2D, and Table 1A and IB compare initial growth upon dilution into media containing various concentrations of ethanol . In the absence of ethanol, KOll appeared to grow slightly better than the mutants both with glucose and with xylose as the fermentable sugar as shown in Figures 2A and 2C. However, KOll was unable to grow in 3.5%(w/w) ethanol while all mutants grew in ethanol concentrations of up to 5% (w/w) .
  • Ethanol tolerance was also compared using other fermentable sugars which may be of interest for fuel ethanol production: lactose, arabinose, and mannose, galactose, sucrose and raffinose.
  • KOll growth was consistently above that of the mutants in the absence of ethanol.
  • KOll failed to grow in ethanol concentrations above 3% (w/w) while the mutants grew in 5% ethanol with all sugars tested.
  • LYOl, LY02 , LY03 , and LY04 were transferred daily on solid medium lacking alcohol for 30 days. Cells were then used to inoculate broth cultures containing ethanol as shown in Figures 2C and 2D. The resulting growth curves were identical to those initially determined.
  • the retention of osmotic tolerance to sugars is essential for the utility of ethanol -tolerant mutants of KOll. As illustrated in Figures 3A and 3B, tolerance to xylose and glucose are similar on a molar basis. With both sugars, KOll appeared more resistant to osmotic stress than the ethanol -resis-tant mutants, although this difference was not dramatic. No differences were observed in growth at 48 °C, the maximum temperature for growth. Both KOll and the mutants grew slowly at this temperature .
  • Plasmid pLOI1531 contains a 3.6 kbp fragment of KOll DNA within the 67.4 min-76.0 min region of the E. coli chromosome (GenBank Accession Number U18997) . Subclones were prepared and sequenced to confirm gene arrangement in KOll and to identify the genes which are responsible for a decrease in ethanol tolerance ( Figure 5) . Only 2 open reading frames were present: crp and a large open reading frame of unidentified function (ORF 0696). A comparison of the effects of pUC18, pLOI1531 and deleted clones (pL0I1532 and pL0I1533) on LY03 indicated that the crp gene (cyclic AMP receptor protein) was responsible for the decrease in ethanol tolerance .
  • pL0I1534 contains a 7.39 kbp fragment of KOll DNA within the 89.2 min -92.8 min segment (Accession Number U00006 of the E. coli chromosome ( Figure 6) .
  • Deleted derivatives of this clone were also sequenced to confirm gene arrangement and revealed the presence of 3 complete and 2 partial open reading frames: dnaB ' , air, tyrB, napA (hobH) , and uvrA ' .
  • a comparison of deleted derivatives identified the air gene (biosynthetic alanine racemase) as being responsible for decreasing the ethanol tolerance of LY02.

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Abstract

Nouveau procédé de sélection permettant d'identifier de nouvelles formes mutantes d'Escherichia coli KO11, une bactérie éthanologène présentant la capacité de croître et de survivre dans ces concentrations d'éthanol supérieures à celles dans lesquelles les Escherichia coli KO11 mères peuvent survivre. Cette nouvelle approche, qui alterne entre la sélection orientée sur la résistance à l'éthanol et la sélection orientée sur la croissance rapide sur un milieu solide présentant un taux élevé de chloramphénicol, a permis d'obtenir des souches potentiellement utiles pour la production d'éthanol.
EP98914390A 1997-04-07 1998-03-31 OBTENTION D'$i(ESCHERICHIA) COLI RESISTANTES A L'ETHANOL HAUTEMENT CONCENTRE Withdrawn EP0973882A1 (fr)

Applications Claiming Priority (3)

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US83490097A 1997-04-07 1997-04-07
US834900 1997-04-07
PCT/US1998/006405 WO1998045425A1 (fr) 1997-04-07 1998-03-31 Obtention d'escherichia coli resistantes a l'ethanol hautement concentre

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EP (1) EP0973882A1 (fr)
JP (1) JP2001518789A (fr)
AU (1) AU6875298A (fr)
CA (1) CA2286334A1 (fr)
WO (1) WO1998045425A1 (fr)
ZA (1) ZA982742B (fr)

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US6102690A (en) 1997-04-07 2000-08-15 Univ. Of Florida Research Foundation, Inc. Recombinant organisms capable of fermenting cellobiose
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ZA982742B (en) 1998-10-05
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CA2286334A1 (fr) 1998-10-15
JP2001518789A (ja) 2001-10-16
AU6875298A (en) 1998-10-30

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