WO2000014268A9 - Modulation du metabolisme microbien et/ou du taux de croissance au moyen d'hormones de croissance vegetales - Google Patents

Modulation du metabolisme microbien et/ou du taux de croissance au moyen d'hormones de croissance vegetales

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Publication number
WO2000014268A9
WO2000014268A9 PCT/US1999/020491 US9920491W WO0014268A9 WO 2000014268 A9 WO2000014268 A9 WO 2000014268A9 US 9920491 W US9920491 W US 9920491W WO 0014268 A9 WO0014268 A9 WO 0014268A9
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Prior art keywords
plant growth
fungi
bacteria
growth hormone
absence
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PCT/US1999/020491
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English (en)
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WO2000014268A1 (fr
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Stephen C Edberg
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Stephen C Edberg
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Priority to AU59110/99A priority Critical patent/AU5911099A/en
Publication of WO2000014268A1 publication Critical patent/WO2000014268A1/fr
Publication of WO2000014268A9 publication Critical patent/WO2000014268A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the invention relates in general to altering bacterial or fungal growth conditions.
  • Bacteria are single cell life forms that do not have a nucleus (Prokaryotes). Fungi are single celled life forms that do have a nucleus. In common they divide, or multiply, by binary fission. Essentially, the body of the microbe increases in size until a signal is generated whereby the cell is split into two. This is accomplished by doubling the genetic material, sequestering each half in opposite sides of the cell, and then dividing the cell into two parts by the laying down of a new cell wall. The cell wall is the physical outer skeleton of microbes. Each cell is called a daughter cell. Bacteria and fungi are not considered plants. However, they share with plants an anatomical structure called the cell wall.
  • the cell walls of microbes and plants are very different physically and chemically - the only commonality is that the cell wall provides external rigid structure to the cells.
  • the cell wall in bacteria, fungi, and plants provide a physically rigid shape to the living entity.
  • the microbe In bacteria and fungi, the microbe is always single-celled. Therefore, the cell wall provides the bacteria or fungi with its shape - e.g., rods, cocci, spiral.
  • the cell wall not only provides shape, but also allows cornpartmentalization.
  • the cell walls of bacteria and fungi are significantly different from those of plants.
  • the growth and differentiation of multi-celled organisms which have specialized structures (e.g., leaves, roots, etc.), are controlled by signals. These signals can be chemical (hormones) or electrical. Signals in protists and fungi have not been described. Each stage in the growth of a plant is controlled by endogenous plant growth regulators which function as hormones. These are known as phytohormones.
  • Plant hormones have the following properties: they are chemically characterized; they show a specific biological activity at low concentrations; they play a fundamental role in regulating plant growth or differentiation; they are generally synthesized at one site and active at another site. Plant growth hormones act as chemical signals to promote growth in plants.
  • a hormone is a molecule generated in one cell that travels to a distant cell where it conveys a message, such as to turn on or to turn off a function.
  • insulin is made in the pancreas that travels to receptors throughout the body to affect the metabolism of glucose.
  • plants contain billions of cells arranged in specialized structures (e.g., roots, branches, phloem, xylem), a growth hormone which is able to act at a distance from the site of production of the hormone is necessary in order to signal plant growth.
  • specialized structures e.g., roots, branches, phloem, xylem
  • Prior art which relates to increasing the yield of endproducts includes: U.S. Patent No. 5,925,807, in which a fungus is used to produce gibberellic acid, with no mention of any effect of GA on the fungus itself; U.S. Patent No. 5,002,603, which discloses a method for stimulating growth of a fungus using isoflavanoids; U.S. Patent No. 5,691,275, which discloses compositions of an alkali metal formononetinate that stimulate the growth of fungi; and U.S. Patent No. 4,911,746, which discloses the use of 1-hydroxylethylenazole compounds to regulate the growth of fungi.
  • the invention includes a method of increasing the metabolic rate of bacteria or fungi, comprising growing the bacteria or fungi in the presence of a first and a second plant growth hormone, wherein the first and second plant growth hormones are different plant growth hormones, and are together present in an amount sufficient to permit an increase in the metabolic rate of bacteria or fungi relative to the metabolic rate of bacteria or fungi grown in the absence of the first and second plant growth hormones.
  • the first plant growth hormone is an auxin and the second plant growth hormone is selected from the group consisting of: gibberellins, cytokinins, absciscic acids, and ethylenes.
  • the invention also includes a method of increasing the metabolic rate of bacteria or fungi, comprising growing the bacteria or fungi in the presence of a plant growth hormone selected from the group consisting of: gibberellins, cytokinins, absciscic acids, and ethylenes, present in an amount sufficient to permit an increase in the metabolic rate of the bacteria or fungi relative to the metabolic rate of the bacteria or fungi grown in the absence of the first and second plant growth hormones.
  • a plant growth hormone selected from the group consisting of: gibberellins, cytokinins, absciscic acids, and ethylenes
  • the invention also includes a method of increasing the metabolic rate of bacteria or fungi to produce a defined product, comprising growing the bacteria or fungi in the presence of a plant growth hormone, in an amount sufficient to permit an increase in the metabolic rate of the bacteria or fungi to produce a defined product relative to the production of a defined product by bacteria or fungi grown in the absence of the plant growth hormone.
  • the invention also includes a method of increasing the growth rate of bacteria or fungi, comprising growing the bacteria or fungi in the presence of a plant growth hormone selected from the group consisting of: gibberellins, cytokinins, absciscic acids, and ethylenes, in an amount sufficient to permit an increase in the rate of growth of the bacteria or fungi relative to the rate of growth of the bacteria or fungi grown in the absence of the plant growth hormone.
  • a plant growth hormone selected from the group consisting of: gibberellins, cytokinins, absciscic acids, and ethylenes
  • the invention also includes a method of increasing the amount of a genetically engineered product produced by a genetically engineered bacterial or fungal cell, comprising growing a genetically engineered bacterial or fungal cell that produces a product of an engineered gene in the presence of an amount of a plant growth hormone sufficient to permit an increase in the amount of the product produced by the cell.
  • the invention also includes a method of decreasing the time for detection of bacteria or fungi in a biological sample, comprising performing a detection step indicating the presence or absence of bacteria or fungi in a sample that is incubating in the presence of an amount of a plant growth hormone sufficient to permit a decrease in the time of incubation relative to the time of incubation of a sample that is incubating in the absence of the plant growth hormone.
  • the invention also includes a method of decreasing the time for detection of bacteria or fungi in a biological sample, comprising incubating the sample in the presence of an amount of a plant growth hormone sufficient to permit a decrease in the time of incubation relative to the time of incubation of a sample that is incubated in the absence of the plant growth hormone, and performing a detection step in which the presence or absence of the bacteria or fungi in the incubated sample is indicated.
  • the invention also includes a method of decreasing the time for determining antibiotic susceptibility of bacteria or fungi, comprising determining the presence or absence of bacteria or fungi that is incubating in the presence of the antibiotic and an amount of a plant growth hormone sufficient to permit a decrease in the time of incubation relative to the time of incubation of bacteria or fungi that is incubated in the absence of the plant growth hormone, the absence of the bacteria or fungi indicating antibiotic susceptibility or the presence of the bacteria or fungi indicating antibiotic resistance.
  • the invention also includes a method of decreasing the time for determining antibiotic susceptibility of bacteria or fungi, comprising incubating bacteria or fungi in the presence of an antibiotic and an amount of a plant growth hormone sufficient to permit a decrease in the time of incubation relative to the time of incubation of bacteria or fungi that is incubated in the absence of the plant growth hormone, and determining the presence or absence of the bacteria or fungi, the absence of the bacteria or fungi indicating antibiotic susceptibility or the presence of the bacteria or fungi indicating antibiotic resistance.
  • the invention also includes a method of decreasing the time for identification of bacteria or fungi in a biological sample, comprising identifying a type of bacteria or fungi in a biological sample that is being grown in the presence of an amount of a plant growth hormone sufficient to permit a decrease in the time of incubation relative to the time of incubation of a bacteria- or fungus-containing sample that is incubated in the absence of the plant growth hormone.
  • the invention also includes a method of decreasing the time for identification of bacteria or fungi in a biological sample, comprising incubating a biological sample suspected of containing a type of bacteria or fungi in the presence of an amount of a plant growth hormone sufficient to permit a decrease in the time of incubation of the bacteria or fungi relative to the time of incubation of the bacteria or fungi that is incubated in the absence of the plant growth hormone, and identifying the type of bacteria or fungi that is present in the sample.
  • the invention also includes a method of decreasing the time required for a diagnostic test involving bacteria or fungi, comprising performing a diagnostic test on bacteria or fungi while growing the bacteria or fungi in the presence of an amount of a plant growth hormone sufficient to permit a decrease in the time required to grow the bacteria or fungi relative to the time required to grow the bacteria or fungi in the absence of the plant growth hormone.
  • the invention also includes a method of decreasing the time required for a diagnostic test for bacteria or fungi, comprising growing the bacteria or fungi in the presence of an amount of plant growth hormone sufficient to permit a decrease in the time required to grow the bacteria or fungi relative to the time required to grow the bacteria or fungi in the absence of the plant growth hormone; and performing a diagnostic test on the bacteria or fungi.
  • the method is performed in the presence of a second plant growth hormone which is different from the plant growth hormone.
  • the method is performed in the presence of a bacterial or fungal resuscitator.
  • the steps preferably include incubating the bacteria or fungi in a medium in the presence of one or more PGHs, and detecting the presence of the bacteria or fungi, or alternatively, determining or measuring the amount of bacteria or fungi in the presence of the PGH. Additional steps may include comparing the amount of bacteria or fungi in the presence of PGH with the amount in the absence of PGH.
  • the invention also encompasses a composition comprising a bacterial or fungal growth medium in combination with a plant growth hormone and a bacterial or fungal resuscitator.
  • the invention also relates to a composition comprising a bacterial or fungal growth medium, a first plant growth hormone, and a second plant growth hormone that is different from the first plant growth hormone, wherein the first and second plant growth hormones are present in the medium in an amount sufficient to increase the growth rate of bacteria or fungi in their absence.
  • the composition further comprises a bacterial or fungal resuscitator.
  • Plant hormones are regulators produced by plants, which in low concentrations, regulate plant physiological processes. Hormones usually move within the plant from a site of production to a site of action. (Committee of the American Society of Plant Pathologists, in Takahashi "Chemistry of Plant Hormones”. CRC press, 1986, page 10).
  • an “amount” of a plant growth hormone (PGH) sufficient to permit a decrease in the time of incubation of bacteria or fungi in the sample relative to the time of incubation of the same type of bacteria or fungi in the absence of the PGH is that amount which permits a statistically significant decrease in incubation time.
  • a “statistically significant decrease” in incubation time may be a 5- 10% decrease or a 25%, 50%, 75%, or 100% decrease in the time of incubation required to reach a given cell density (as measured, for example, by optical density (O.D.)).
  • an “amount” in this context may be in the range of about 0.001-10 mm of PGH, for example, in a subrange of 0.01-10mm, or 0.1-1 mm.
  • a "detection step" indicating the presence or absence of bacteria or fungi in a sample may be the detection of the density of bacteria or fungi in the sample, e.g., including but not limited to counting of bacterial or fungal colonies within a given area of a solid agar; detection of a fluorescent signal generated by the bacteria or fungi; detection of a metabolic product of the bacteria or fungi; release of 14 CO 2 from a radiolabeled substrate such as a metabolic product of the bacteria or fungi; and cytometry (cell counting) or O.D. determination.
  • incubating may encompass incubating in liquid or solid or semi-solid (e.g., agar) medium, and therefore includes such phrases as “being grown in” or being grown on” or “cultured in” or “cultured on”.
  • “Decreasing the time for detection" of bacteria or fungi in a sample refers to a decrease in time between initiating and terminating a given assay. According to the invention, such a decrease may be at least 10% (hours, minutes, or units of time), and also may be more such as 20-50%, or even 75-95%.
  • the time for detection of bacteria or fungi in a blood culture usually requires an average of 36 hours to obtain a positive signal.
  • the invention provides for a decrease of at least about 3.6 hours and even more, e.g., 4.0 hours, 5.0 hours, 8 hours, or more.
  • the invention provides for a decrease time to detect the bacteria from the norm of about 22 hours to the improved time of 20 hours; such a decrease therefore may be at least 2 hours, if not by 3 hours, 4 hours, 5 hours or more.
  • “Decreasing the time for determining” a given result refers to a decrease in time (minutes, hours, days, etc.), between initiation of the assay (for example, addition of the sample suspected of containing bacteria) and termination of the assay by performing the detection, for example, if the time for determining antibiotic susceptibility is normally about 24 hours, then a decrease according to the invention may be to about 21.6 hours, or to 19.2 hours, or to 16.8 hours. Alternatively, where the result determined is, for example, the activity of an enzyme for fermentation of a sugar, then the time for determining enzyme activity may be decreased from about 5 hours to about 4.5 hours, or 4 hours, 3.5 hours or 3 hours.
  • Antibiotic susceptibility refers to the lowest concentration of antibiotic at which the microbe's ability to multiply is significantly inhibited (i.e., cell doubling is >90% inhibited in a 24 hour period).
  • Antibiotic resistance refers to the ability of a microbe to multiply in the presence of clinically useful concentrations of antibiotic.
  • Incubating and “determining” or “performing” when used in the present tense and together refer to coordinated steps in which one process is performed while the other also is actively performed. In contrast, “incubated” indicates, as used in the past tense, that the step is completed.
  • an "antibiotic” is defined herein as a chemical (whether an isolated natural chemical or a synthetic chemical) that inhibits the growth and/or multiplication of a microbe, and may be present in a given medium in the range of about 0.5 ⁇ g/ml to several hundred (200) ⁇ g/ml.
  • Antibiotics useful in the invention include but are not limited to those listed in Table 7.
  • Identifying a type of bacteria or fungi refers to assigning to a microbe a useful and reproducible identifier.
  • This identifier may be a genus and species name (e.g., Enterococcus faecalis), a genus name only (e.g., Enterococcus), an antigen identifier (e.g.. Group A streptococcus), or a set of phenotypic characteristics.
  • a "type” of bacteria or fungi refers to the identifier selected.
  • “decreasing" the time for identification of bacteria or fungi refers to a decrease in time that permits assignment of an identifier.
  • “Growing” bacteria or fungi refers to incubating bacteria or fungi under conditions sufficient to permit doubling of the bacteria or fungi in a given time period, that is, an increase in the number of bacteria or fungi over time.
  • “Growth rate” or “rate of growth” refers to the doubling time of bacteria or fungi, i.e., the time period in which a given number of bacteria or fungi doubles; for example, E.coli may grow at a rate of about 1 doubling every 20 minutes under conventional growth conditions at 37 °C.
  • Conventional growth rates for the following bacteria are as follows (Bacterium, Optimal Generation Time, Average Generation Time): E. coli, 20 minutes, 30 minutes; Enterococcus, 30 minutes, 40 minutes; Pseudomonas aeruginosa, 30 minutes, 40 minutes; Salmonella typhimurium, 20 minutes, 40 minutes; Mycobacterium tuberculosis, 2 hours, 6 hours.
  • a "diagnostic test” refers to an assay in which a type of a microbe is identified or the presence of a microbe is determined. Some diagnostic tests do not require growth of the microbe, for example a test for production of a constitutive enzyme, where the assay time is reduced according to the invention from an average of 5 hours to an average of 4 hours; production of an inducible enzyme, e.g., where the assay time is reduced according to the invention from an average of 22 hours of incubation to an average of 20 hours of incubation.
  • a “genetically engineered bacterial cell” or a “genetically engineered fungal cell” refers to a host bacterial or fungal cell containing a recombinant or genetically engineered DNA.
  • a “product” of an “engineered gene” refers to an RNA or a protein.
  • a “defined product” may include a direct product ⁇ RNA, protein, or an indirect product, i.e., altering a regulatory gene or an upstream pathway gene for production of a defined product, or a pharmaceutical, or commercially useful chemical compound.
  • an “increase” in the amount of product produced by a cell refers to an increase by 5- 10% or more (e.g., 25%, 50%, 75%, 100% or more), or two-fold or more (e.g. 5-fold, 10- fold, 50-fold or more) of the product. Percentages refer to w/w or w/vol. Further features and advantages of the invention will become more fully apparent in the following description of the embodiments thereof, and from the claims. DESCRIPTION
  • a microbe that is, a bacterium or fungus
  • a microbe may be directed to produce more of itself (i.e., to decrease the amount of time required to divide from one bacterium or fungal cell into two) in a shorter time period by incubating the bacteria or fungi in a medium that contains at least the minimal nutritional requirements for bacterial or fungal growth and also contains a plant growth hormone that is selected from the group consisting of: gibberellins; cytokinins; abscisic acids; and ethylenes.
  • the invention also is based on the discovery that a microbe may be directed to produce more of itself in a shorter time period by incubating the bacteria or fungi in a medium that contains at least two different plant growth hormones.
  • the invention also is based on the discovery that a microbe may be directed to increase its metabolism. Although it is possible that an increase in metabolism leads to a decrease in the generation time, this latter aspect of the invention relates to a general increase in metabolism and also an increase in metabolism with reference to increasing production of a defined product, which is distinct from and does not necessarily involve a general increase in metabolism. An increase in metabolism that increases production of a defined product preferably does not also involve an increase in growth rate, at least above a minimal level of 5-10%.
  • Treatments or factors leading to a decrease in the generation time of bacteria or fungi may be useful to include in media used to test sterility, such as for pharmaceuticals, or in the culture of blood for sepsis.
  • An increase in metabolism would be useful, for example, if one wanted to use the microbe to produce an endproduct.
  • the invention thus also is based on the discovery that the time required to perform a diagnostic or antibiotic susceptibility test or to produce a genetically engineered product of a bacterium or fungus may be significantly reduced by growth of the bacteria or fungi in a medium containing a plant hormone.
  • the invention thus also is based on the discovery that the above assays may be performed in the presence of a bacterial or fungal resuscitator and therefore media according to the invention also may include a bacterial resuscitator.
  • Bacteria and Fungi Useful According to the Invention A. Bacteria Useful According to the Invention:
  • a "bacterium” is herein defined as a protist (without an organized nucleus) that divides by binary fission. Classes and types of bacteria useful according to the invention are presented in Table 1.
  • Fungi are a diverse group of eukaryotes, which are currently undergoing taxonomic redefinition from parameters based primarily on phenotypic characteristics to those based on genotypic characteristics.
  • the nomenclature of fungi is governed by the International Code of Botanical Nomenclature (ICBN) as adopted by each International Botanical Congress. (Greuter, W, et. al. 1994. International code of botanical nomenclature (Tokyo Code) adopted by the Fifteenth International Botanical Congress, Yokohama, August- September 1993. Rengnum Veg. 131.
  • Table 3 presents an overview of yeast general from each of the four classes of fungi presented in Table 2.
  • Genera (Ascmycotina) (Deuteromycotina) Genera Genera
  • Botryoascus ( 1 ) Myxozyma (9! Filobasidium (5) Kockovaella (2) Cephaoloascus (2) Oosporidium (1 ) Leucosporidium (3) Kurtzmanomyces (2)
  • Rhodosporidium Rhodotorula
  • Cyniclomyces ( 1 ) Trigonopsis (1 ) Sporidiobolus (3) Sporobolomyces (27) Debaryomyces (10) Sterigmatosporidium ( 1 ) Sterigmatomyces (2)
  • Lodderomyces ( 1 )
  • Saccharomycodes (2) Saccharomycopsis (6)
  • the Yeasts A Taxonomic Study (edited by C.P. Kurtzman and J.W. Fell; published by Elsevier 1997) .
  • An alternative means of classifying the fungi is to examine their placement in the three major kingdoms of life: Table 4. Phyla and classes of the three kingdoms where fungi are placed.
  • Fungi have been utilized for thousands of years for the commercial production foods and medicines. Even before it was possible to isolate fungi in culture, a subculture of a volume from a successful "ferment” was transferred to a starting material to initiate fermentation. Examples include yogurts, pickles, tofu, and vinegar. Henle first described what became known as the Henle-Koch postulates for disease causality by studying the production of beer by yeast. Likewise, the uncontrolled growth of fungi could result in the spoilage of foods and ferments. Pasteur's early work was conducted, and funded, by the wine and beer industries in France, and resulted in the process today we know as Pasteurization. Some commercially important and common examples of yeast (that is, fungal) fermentations are presented in Table 5, below.
  • Fungi being eukaryotic cells, have proven efficient at producing molecules that are biologically active in humans. For example, insulin and human growth hormone are produced in this way.
  • Saccharomyces cerevisiae is described as a fungus that is commonly used to produce natural and genetically engineered commercial products. Saccharomyces cerevisiae is one fungal species that has been utilized for centuries to produce commercially important products such as bread and fermented products. More recently, plasmids which incorporate the coding sequences to produce commercially useful proteins and other endproducts have been introduced into Saccharomyces cerevisiae. The fungus can be grown in artificial culture media to produce endproducts.
  • the artificial culture media are particularly useful in the production of genetically engineered endproducts because the media constituents, being simple molecules, are readily separated from the complex genetically engineered endproducts.
  • the basic requirements for Saccharomyces cerevisiae, and most yeasts, include 2 Ingredient Source Concentration Range
  • Carbon Simple sugar such as glucose 5-20 g/liter Hydrogen Protons from acidic environments pH5-7 Oxygen Air, added 02 Nitrogen Ammonium ions (such as from 3-20 g/liter ammonium sulfate, urea, amino acids)
  • This invention can modify the growth and/or metabolism rate of fungi to increase the efficiency of production of commercial useful endproducts. Moreover, by increasing the growth and/or metabolism of fungi, the invention can increase the efficiency of producing products that require fungi to be present. Moreover, the invention can decrease the time needed to finalize the endproduct, whether the end product is a molecule or food. Fungi can also be pathogenic in humans. Their diagnosis follows the same principles as bacterial diagnosis: isolation from a specimen; detection of an antigen; detection of an antibody. After isolation, anti-fungal antibiotic susceptibility tests should be performed. The invention will make all of these steps more efficient.
  • the invention is practiced on fungi to enhance their growth rate and/or metabolism.
  • Plant growth hormones in combination with each other, with or without resuscitators, are incorporated into fungal growth media.
  • the media may be either liquid or solid.
  • the combinations of PGHs with or without resuscitators may be included in the growth and/or metabolism medium.
  • I AA or heteroauxin, the broader term
  • mixed giberellic acids, and zeatin or kinetic riboside alone or in combination
  • Combinations can be with INAxGAs; IAAxKinetins;GAsxkinetins; IAAxGAsxkinetins.
  • a resuscitator as described herein may additionally be added to a culture containing PGHs in order to further enhance the fungal growth.
  • Proportions will be specific to individual species of fungi and determined by the reaction of the target fungi. To identify the proportions of one or more PGHs and/or resuscitators effective to enhance the growth or metabolism of a particular fungus, a checkerboard as described below in "Combinations of PGHs Useful According to the Invention" is employed.
  • Fungal growth detection is enhanced according to the invention if there is a statistically significant decrease in the time required for signal perception in the presence of one or more PGHs relative to the time required to detect growth in the absence of PGHs.
  • Fungal metabolism is enhanced according to the invention if there is a 10% or greater increase in a measured metabolic indicator in the presence of one or more PGHs relative to the indicator in the absence of PGH.
  • Antibiotics useful according to the invention include, but are not limited to those listed in Table 7.
  • Antibiotic (mcg/mL) (mcg/mL) ampicillin 4 32 mezlocillin 8 128 amoxicillin/clavulanic acid 2/1 32/16 ticarcillin/clavulanic acid 8/1 128/2 cafazolin 4 64 cephalothin 4 64 cefuroxime 4 64 cefepime 4 64 cefoxitin 4 32 ceftazidime 4 64 ceftriaxone 4 128 cefixime 0.5 8 imipenem 2 32 aztreonam 4 64 gentamicin 2 16 amikacin 8 64 tetracyline 2 32 ciprofloxicin 0.5 8 trimethoprim/sulfamethoxazole 1/19 8/152 chloramphenicol 4 64 sulfonamides 50 400 trimethoprim 2 32
  • Plant growth hormone a general term to mean a regulatory chemical of plant and plant cell growth. These are associated with a wide variety of physiological functions in plants, and are also known as phytohormones or plant growth regulators.
  • Plant growth hormones are naturally plant products, but can be made to be produced, via recombinant DNA techniques, by fungi and bacteria.
  • Chemical classes of plant growth regulators useful according to the invention are as follows: auxins; gibberellins; cytokinins; abscisic acids; and ethylenes.
  • Plant growth hormones include the following classes: Auxins:
  • auxin is a general term for compounds whose primary activity is to induce elongation in shoot cells. They resemble indole-3-acetic acid (IAA) in physiological action.
  • IAA indole-3-acetic acid
  • Auxins include but are not limited to the following: centrophenoxine p-chlorophenoxyacetic acid chlorogenic acid trans-cinnamic acid
  • Gibberellins include but are not limited to the following: gibberellic acid gibberellin A3 gibberellin A4 gibberellin A7 giggerellin A13 iso-gibberellin A7 iso-gibberellin A7 methyl ester
  • Cytokinins have been shown to control apical dominance, promote leaf and cotyledon growth, control maintainance of chlorophyll in excised leaves, promote seed germination, and promote cell-division in tissue cultures systems. They participate in the control of development and senescence of plants.
  • the first discovery of a cytokinin was made by Jabotinkski and Skoog et al., using tobacco tissue culture techniques. Cytokinins are purine (substituted adenine) derivatives. Zeatin and Kinetin are examples.
  • Cytokinins include but are not limited to the following: adenine adenine hemisulfate
  • Ethylene is a gaseous substance that has growth regulating effects in higher plants.
  • Abscisic acids and other plant growth hormones include but are not limited to the following: ciSjtrans-abscisic acid ancymidol chlorocholine chloride
  • Combinations of PGHs Useful According to the Invention From 0.001 to 10 mmol (millimolar) final concentration of IAA (or heteroauxin, the broader term), mixed giberellic acids, and zeatin or kinetic riboside alone or in combination. Combinations can be with IAAxGAs; IAAxKinetins;GAsxkinetins; IAAxGAsxkinetins. Any combination of PGHs, which includes any combination from all five classes of PGHs, is useful according to the invention. Proportions of PGHs will be particular to the specific microbe and determined by the reaction of the target microbe(s).
  • a "checkerboard" titration is instituted in which all combinations of two given series of concentrations of two PGHs are prepared and examined for their effect on bacterial or fungal growth and/or metabolism. For example, sequential one-half dilutions of a first PGH (PGH#1) from an initial concentration of 10 mmol to a final concentration of 0.001 mmol are prepared on the X axis of the checkerboard, and sequential one-half dilutions of a second PGH (PGH#2) from an initial concentration of 10 mmol to a final concentration of 0.001 mmol are prepared on the Y axis.
  • PGH#1 sequential one-half dilutions of a first PGH (PGH#1) from an initial concentration of 10 mmol to a final concentration of 0.001 mmol are prepared on the X axis of the checkerboard
  • each axis will have concentrations of 10, 5, 2.5, 1.25, 0.625, 0.31, 0.15, 0.075, 0.037, 0.018, 0.09, 0.045, 0.0225, 0.0011, 0.0005, 0.00025, 0.000125, and 0.0000625 mmol.
  • test tubes that fill in the checkerboard cover the range of combinations of PGH#1 and PGH#2 useful in the invention.
  • this checkerboard pattern is reproduced 18 times.
  • a concentration of PGH#3 of 10, 5, 2.5, 1.25, 0.625, 0.31, 0.15, 0.075, 0.037, 0.018, 0.09, 0.045, 0.0225, 0.0011, 0.0005, 0.00025, 0.000125, 0.0000625 mmol is added.
  • Control cultures without PGHs are also established. See Table 8 for exemplification of a method of determining a synergistic effect using combinations of PGHs.
  • the checkerboard approach as described herein permits testing combinations of PGHs alone, in combinations of two, and in combination of three, for their ability to exert an effect on bacterial or fungal cell growth.
  • the samples containing bacteria or fungi are incubated and monitored over time for either the density of bacteria or fungi (for example, by O.D. measurement) or for the amount of an indicator product (such as ,4 CO 2 ) or metabolic end product (such as glucose isomerase, or a product of genetic engineering, such as insulin or urokinase).
  • a similar approach may be taken to determine the optimal amount of a particular resuscitator or resuscitators to further enhance the growth or metabolism of a fungal culture.
  • a fungal growth medium containing an optimal level of one or more PGHs, as determined by a prior checkerboard-type titration may be supplemented with varying concentrations of one or more resuscitators in a subsequent checkerboard-type titration, allowing the identification of the optimal concentration(s) of resuscitator(s).
  • the basic formulation for bacterial growth medium containing one or more PGHs is to add an amount of PGHs (one or more than one PGH as a mixture) in the range of 0.001 to 10 mmol (millimole) final concentration, e.g., in the range of 0.01 to 10 mmol, or in the range of 0.1 mmol to 1 mmol.
  • the invention contemplates the addition of one or more PGHs to a bacterial growth medium, wherein the single PGH or any combination of PGHs has the effect of increasing the growth rate or metabolic rate of the bacteria in the growth medium.
  • bacterial culture media to which PGHs may be added include (mass amounts of the various components are per liter of medium): Columbia broth fused for the culture of bloods Polypeptone 10.0 grams
  • the PGHs at concentrations of 10 ppm are sterile filtered and added to the medium after autoclaving. Also after autoclaving, a carbohydrate, such as lactose is added to a final concentration of 1 percent.
  • the agar is dispensed into test tubes and allowed to harden. The top of a colony is touched with a straight bacterial wire, and stabbed into the CTA agar. Fermentation is indicated by a change in color from red to yellow.
  • fungi will grow in bacterial growth media as described above or as known in the art. Obviously, those media which contain an anti-fungal agent, such as MacConkey's agar, will not be useful for fungal growth. In addition to media originally optimized for bacterial cell growth, media optimized for fungal growth may be useful. Examples of media optimized for fungal growth are presented in Table 9.
  • fungal media include, but are not limited to Sabouraud's broth, corn meal broth, malt extract broth, malt extract dextrose broth, and yeast extract broth. All of these media are available from Difco.
  • the liquid fungal growth media can be made as solid medium with the addition of approximately 20 grams of agar per liter to the final mixture.
  • Plant growth hormones of the same types and in the same range of concentrations described herein for enhancing bacterial growth and/or metabolism are useful for enhancing fungal growth and/or metabolism. That is, a PGH, alone or in combination with another PGH of a different class or a different type in the same class, can be used in a wide variety of fungal growth media formulations.
  • the basic formulation for fungal growth medium containing one or more PGHs is to add an amount of PGHs (one or more than one PGH as a mixture) in the range of 0.001 to 10 mmol (millimole) final concentration, e.g., in the range of 0.01 to 10 mmol, or in the range of 0.1 mmol to 1 mmol.
  • the invention contemplates the addition of one or more PGHs to a fungal growth medium, wherein the single PGH or any combination of PGHs has the effect of increasing the growth rate or metabolic rate of the fungi in the growth medium.
  • resuscitators compounds which help microbes metabolize more efficiently.
  • resuscitators compounds which help distressed microbes recover, or microbes metabolize more efficiently.
  • resuscitators are compounds either made by the microbe or obtained from the environment. Generally, they are naturally occurring, although chemical analogs may be effective. Resuscitators may assist in the metabolism or recovery of both bacteria and fungi.
  • resuscitators useful according to the invention include but are not limited to [% or units referred to are in final concentrations in media]: intermediary metabolites such as sodium pyruvate: 0.001 to 1.0% catalase: 100 to 4000 Units acetic acid: 0.001 to 0.1% water soluble vitamins, particularly the B vitamin group:0.001 to 1.0% hemin: 0.001 to 0.01% and combinations of the above.
  • Methods and compositions of the invention may direct a microbe into producing more of itself (i.e., decrease in the generation time) or may direct the microbe into increasing its metabolism. Often, these processes are linked; an increase in metabolism leads to a decrease in the generation time. However, the processes may not be linked.
  • a factor or combination of factors leading to a decrease in generation time may be useful to include in media used to test sterility, such as for pharmaceuticals, or in the culture of blood for sepsis. An increase in metabolism would be useful, for example, if one wanted to use the microbe to produce an end product.
  • PGHs plant growth hormones
  • a decrease in the time required for a diagnostic test is effected as follows. 1. Blood Culture When a patient develops fever and is either in, or admitted to, a hospital it is routine to do a "fever workup". The fever workup almost always includes obtaining blood and inoculating that blood in microbial growth medium. The physician wishes to establish if the patient has sepsis, which is serious and can also result in the seeding of organs. In effect, a sterility test of the patient's blood is performed. Typically, blood from the patient is inoculated into liquid growth medium. The liquid growth medium is incubated at 35-37° C. Periodically it is examined for evidence of microbial activity.
  • Microbial activity may be monitored by: visual observation (or by examining changes in optical absorbance); monitoring for the production of l4 CO 2 ; or by the change in a fluorescent indicator attached to the bottle holding the growth medium. Once evidence of microbial activity is noted, a sample from the blood culture bottle is removed and analyzed to determine which type of microbe is present (e.g., bacterium or fungus, gram positive or gram negative). The physician is notified by telephone, since microbial growth is considered a medical emergency.
  • type of microbe e.g., bacterium or fungus, gram positive or gram negative
  • one or more PGHs are added to the blood culture medium. For example, from 0.001 to 10 mmol [millimole] final concentration of IAA alone, and with 0.001 to 10 mmol mixed gibberellic acids, and with 0.001 mmol of kinetic riboside, is added to the blood culture bottles. These are compared with samples to which blood sample, but no PGHs are added. Patients are sampled so that there are 50 with sepsis. In the bottles with the PGH(s), there is a statistically significant reduction of the average time of detection of microbial growth.
  • a microbial antigen is either a protein, polysaccharide, or combination of these that will elicit an immune response in an animal.
  • One way the animal responds is to produce proteins called antibodies.
  • the antibodies combine with the antigen so that the animal may more rapidly eliminate the microbe exhibiting the antigen from its body.
  • Antigens are specific for particular microbes. For example, if one immunizes an animal, such as a rabbit or goat, with a microbe, and then harvests the animal's serum, that serum will contain antibody to that microbe, and only that microbe. The use of antibodies in the laboratory is called serology.
  • antigens are useful, and economically important, in a number of fields including, but not limited to production of vaccines (antigens are purified and inoculated into people to produce antibody - for example, tetanus toxoid, diphtheria toxoid, meningitis vaccine, and Hemophilus influenzae b [Hib]), and the identification of bacteria or fungi in the climcal laboratory (a bacterial or fungal colony is mixed with antisera specific for a variety of bacteria or fungi; if there is a reaction with one, the bacterium or fungus is identified).
  • vaccines antigens are purified and inoculated into people to produce antibody - for example, tetanus toxoid, diphtheria toxoid, meningitis vaccine, and Hemophilus influenzae b [Hib]
  • identification of bacteria or fungi in the climcal laboratory a bacterial or fungal colony is mixed with antisera specific for
  • PGHs are added to the liquid culture medium. For example, from 0.001 to 10 mmol [millimole] final concentration of IAA alone, and with 0.001 to 10 mmol mixed gibberellic acids, and with 0.001 mmol of kinetin riboside or zeatin, is added to the liquid culture medium.
  • the optimal concentrations of PGH(s) is determined, for example, by performing a "checkerboard" titration as described in the section "Combinations of PGHs Useful According to the Invention".
  • the microbe is grown in a chemostat, in which fresh medium is slowly pumped into a vessel, and an equal amount of spent culture medium is simultaneously removed. With the spent culture medium is the microbe, and the antigen. The more antigen leaving with the spent medium, the more commercially viable the process. A comparison of chemostats with and without PGHs will show at least a 10% increase in antigen yield.
  • a genetically engineered bacterium or fungus produces a useful product.
  • the clone grows in liquid medium, generally in a chemostat type device (new medium is added, old medium with the desired product is removed). In this case, one wishes to increase the metabolism of the clone, not its numbers.
  • the addition of one or more plant growth hormones increases the end product by at least 5%.
  • a bacterium or fungal cell which is changed by manipulating its DNA is called "genetically engineered”. These bacteria or fungi are constructed by inserting plasmids or genetic elements that code for the production of a particular commercial product into the chromosome. Alternatively, natural bacteria or fungi that make small amounts of the desired endproduct are manipulated to produce more. They may be subjected to ultraviolet light or mutagens.
  • An example of a commercially important product produced from a genetically engineered bacterium is Glucose Isomerase (GI). This enzyme catalyzes the conversion of glucose to fructose. Fructose is the major sugar used in food processing.
  • D-Glucose/xylose isomerase (D-xylose ketol isomerase; EC 5.3.1.5), commonly referred to as glucose isomerase (GI), is one of the three highest tonnage value enzymes, amylase and protease being the other two. It catalyzes the reversible isomerization of D- glucose and D-xylose to D-fructose and D-xylulose, respectively. Isomerization of glucose to fructose is of commercial importance in the production of high-fructose corn syrup. GI catalyzes the isomerization of both glucose and xylose.
  • the yields of GI from various potent producer organisms range from 1,000 to 35,000 U liter "1 . Further improvement in the yield and the properties of the enzyme may be achieved by strain improvement, using either conventional mutagenesis or recombinant DNA technology. Further improvement of yield is obtained according to the invention using conventional growth media and a combination of PGHs.
  • GI is generally produced by submerged aerated fermentation.
  • GI is generally an intracellular enzyme, except in a few cases when the enzyme production is extracellular.
  • the enzyme is extracted from microbial cells by mechanical disruption (such as sonication, grinding, or homogenization) or by lysis of the cells with lysozyme, cationic detergents, toluene, etc. (Chen, W.P. 1980. Glucose isomerase. Process Biochem. 15(June/July):30-35).
  • a plant growth hormone is sterile filtered and added to the medium in the chemostat at a concentration of about 10 ppm for each plant growth hormone added. If more than one plant growth hormone is added, then each plant growth hormone should be present at about at least 10 ppm in the medium.
  • Culture medium may be prepared as follows (amounts are per liter of medium): starch lOg; potassium nitrate 2g; sodium chloride 2g; magnesium sulfate 0.05g; calcium carbonate 2g; ferric sulfate 0.0 lg; in 10 mmol Hepes buffer made in distilled water. After mixing, 2 grams of agar is added and autoclaved. To this medium is added 10 grams of filter sterilized glucose. If one wishes to exclude fungi, 100 mcg/mL of nystatin is added.
  • Microbial isolates, soil sample, etc. are plated on the surface of the agar. Colonies are harvested after 24 hours. GI is assayed from the cells by assaying for the production of fructose (Kulka, RG. 1956. Colorimetric estimation of ketopentoses and ketohexoses. Biochem. 1 63:542-548).
  • PGHs are added to the liquid culture medium.
  • concentration of IAA alone from 0.001 to 10 mmol [millimole] final concentration of IAA alone, and with
  • the following example describes growth of bacteria using a combination of resuscitators and PGHs for the recovery of chlorine damage coliforms from drinking water.
  • Clorox (5.25% sodium hypochlorite) is diluted to 2.0 mg of chlorine per liter.
  • a coliform isolate is inoculated into tryptic soy broth and incubated for 4 hours at 24°C. It is diluted to a concentration of 10 9 per mL.
  • Equal amounts of sodium hypochlorite and bacterial growth medium are mixed. After 6 minutes, subcultures are made onto nutrient agar plates. In parallel, the same protocol is followed with the substitution of distilled water for sodium hypochlorite, to serve as a control.
  • E. coli ATCC 25922 was grown overnight in trypticase soy broth (TSB) and diluted in normal saline using a spectrophtometer absorption to 10 5 /mL. Colony counts were made on MacConkey's agar using quantitative loops of volume 0.01 mL on one half of the plate and 0.001 mL on the other side of the plate. Mixed Giberillic acids were obtained from Sigma. The mixture was first dissolved in ethanol and then subsequently diluted in distilled water.
  • TSB milligrams per liter
  • TDB Trypticase soy broth
  • mixed gibberellic acids were added to produce final concentrations of 50; 6.25; 0.75 mcg/mL; and a negative control lacking gibberellic acids.
  • Incubation was at 35 °C.
  • TSB tubes were placed in a Spectronic 20 spectrophotometer and O.D. measured at 560 nm.
  • n generation time difference
  • N turbidity value
  • Klebsiella pneumoniae was isolated from a drinking water sample. It was grown overnight in trypticase soy broth (TSB) and diluted in normal saline to lOVmL using spectrophotometer absorption. Colony counts were made on MacConkey's agar using quantitative loops of volume 0.01 mL on one half of the plate and 0.001 mL on the other side of the plate.
  • TTB trypticase soy broth
  • Zeatin was obtained from Sigma. It was first dissolved in ethanol and then subsequently diluted in distilled water. Experiment: Concentrations of Zeatin were made at 100; 50; 25; 12.5; 6.25; 3.1; 1.5;
  • TSB Trypticase soy broth
  • O.D. Optical Density
  • the generation time of K pneumoniae is expected to be significantly less in the presence of Zeatin than without it.
  • DM Defined Medium
  • the base medium consisted of ammonium sulfate (5 grams/Liter) as the nitrogen source; glucose (5 grams/Liter); calcium chloride (10 mg/Liter); and magnesium sulfate (10 mg/Liter).
  • DM was made with the following additives: 20 mg/L of mixed gibberellins + 20 mg/n L kinetin (GK); 50 mg/1 of IAA + 20 mg/L of mixed gibberellins (IG); and 20/20 mg/L mixed gibberellin+kinetin+50mg/L IAA (GKI).
  • the generation time of E. coli was significantly less in the presence of PGHs than without it.
  • n log N2-logNl 0.301
  • n generation time difference
  • N turbidity value
  • the base medium consisted of trypticase soy broth (TSB). To this broth, kinetin was added at a concentration of 10 mg/Liter and 20 mg/Liter and mixed Gibberellic acids at 10 mg/Liter and 20 mg/Liter. A control lacking PGHs was also inoculated. Incubation was at 35 °C. TSB tubes were placed in a Spectronic 20 spectrophotometer and O.D. was measured at 560 nm.
  • the generation time of E. coli was significantly less in the presence of mixed phytohormones than without it.
  • Bacteria and other microbes metabolize in addition to growing (multiplying). Fermentation of sugars is one of the primary metabolic activities of many microbes. Accordingly, the activity of phytohormones on the ability of Escherichia coli to ferment sugars was examined. Two sugars were studied. First, glucose was examined since it is the central carbon/energy source molecule. In addition, sorbitol (an alcohol sugar) was studied since E. coli is a "slow" fermenter of this.
  • Phytohormones included IAA, kinetin, and mixed gibberellin.
  • E. coli was ATCC 25922.
  • Glucose was made at 5 grams/liter into 5 grams/liter of proteose peptone #3 (does not contain sugar) to which 0.2% of bromocresol purple was added.
  • Bromocresol purple is blue/purple at a basic pH and turns yellow in the presence of the acid produced by the fermentation of sugar.
  • Sorbitol at 5 grams/liter was made in the same pp#3 solution.
  • Test media were made with the following additives:
  • the top of a single colony of the test bacteria was touched with a standard bacteriological transfer wire and the bacterial inoculum transferred to 1 ml of each of the substrates. Incubation was at 35 °C. Each hour the color of the tubes was recorded.
  • AST antibiotic susceptibility test
  • the basic principle of the AST is that the test bacterium is inoculated into culture medium with and without antibiotic. After incubation, the growth of the bacterium in both culture media is compared. If growth in the presence of the antibiotic is significantly less compared to growth observed in culture medium without antibiotic, the test bacterium is considered susceptible to that antibiotic. If the growth of the test bacterium in the presence of the antibiotic is not significantly different from growth in the culture medium without antibiotic, the test bacterium is considered resistant.
  • resistant and susceptible are transmitted to the patient's physician and the physician will use that information to include or exclude that antibiotic from the treatment choices. Accordingly, the more rapidly the AST can be performed, the better the patient's care. The accuracy of ASTs is further controlled because the FDA approves all methods.
  • test bacterium in culture medium There are a number of ways known in the art to measure the growth of the test bacterium in culture medium. Examples include measuring turbidity in liquid medium or on solid agar; measuring metabolism of a vital dye such as resazurin or tetrazolium salts; measuring production of acid from a sugar, etc.
  • Phytohormones used in this example included IAA, kinetin, and mixed gibberellins.
  • test bacteria were E. coli ATCC 25922 and Staphyloccocus aureus ATCC 25923.
  • Antibiotic Susceptibility test The antibiotic susceptibility test was conducted in FDA approved Mueller-Hinton broth (MHB). Resazurin (as Alamar Blue, from Accumed Int'l) was added to produce a 10% solution (MHBR). The antibiotic gentamicin (gm) was added to MHBR to produce a final concentration of 4 mcg/mL (MHBRgm).
  • MHBR and MHBRgm were separately dispensed into 12x75mm test tubes, 1 mL per test tube.
  • MHBR and MHBRgm was made with the following additives: 20 mg/L of mixed gibberllins + 20 mg/mL kinetin; 50 mg/L of IAA; and 20/20 mg/L mixed gibberellins+kinetin+50mg/L IAA.
  • EXAMPLE 14 This experiment is the same as that described in Example 10, with the substitution of the commercial Colilert ® test medium for the defined medium.
  • the Colilert ® test is itself a defined medium, and an iteration of the Defined Substrate Technology ® .
  • the test bacteria were: Enterobacter cloacae isolated from the environment and Escherichia coli ATCC 25922.
  • E. cloacae is a total coliform and turns the Colilert ® test yellow.
  • Escherichia coli is a total coliform that turns the Colilert ® test yellow (total coliform part of the test) and fluorescent (E. coli part of the test).
  • test bacteria were diluted using a Spectronic 20 to a concentration of 10 bacteria mL.
  • Fungi e.g., Saccharomyces cerevisiae, which are genetically engineered to produce urokinase, may be made as follows.
  • Urokinase is a mammalian protein that plays a central role in the blood-clotting mechanism. It is administered to patients to dissolve blood clots. Urokinase has separate sites responsible for binding to the blood clot and protease activity to dissolve the blood clot.
  • a clone o ⁇ Saccharomyces cerevisiae constructed by introducing a plasmid that codes for the following characteristics: BamHICUP 1 promoter site; ampicillin resistance; urokinase coding sequence; ubiquitin regulator genes; and G protein recognition genes is used (see Ecker, D.J. et al., 1989. Increasing gene expression in yeast by fusion to ubiquitin. J. Biol. Chem. 264: 7715-7719.).
  • the Saccharomyces cerevisiae strain bearing the urokinase plasmid is grown in synthetic medium as described above, with the addition of 100 uM CuSO 4 to induce expression. After shaking for 2 to 3 hours at 30°C, the Saccharomyces cerevisiae cells are centrifuged, washed, and resuspended in buffer to lyse them (50mM Tris, pH 7.6 with 10 mM dithiothreitol (a reducing agent) and 1% sodium dodecyl sulfate (SDS, a detergent)).
  • the active urokinase can be isolated by removing the salts from the proteins, by dialysis, or other means that separate proteins from salts.
  • the production of urokinase by genetically altered yeast cells may be enhanced by adding one or more PGHs, with or without a resuscitator, to the culture medium in a concentration ranging from 0.001 to 10 mmol.
  • concentration and combination of PGHs effective to enhance the production of urokinase may be determined through titrations as described herein in the section "Combinations of PGHs Useful According to the Invention".
  • Enhancement of urokinase production is determined by comparison of the amount of urokinase produced by cells grown in the absence of PGHs and/or resuscitators with the amount produced by cells grown under the same conditions and for the same amount of time but in the presence of PGHs and/or resuscitators.
  • a statistically significant increase, preferably 10% or more, in the amount of urokinase produced in the presence of PGHs and/or resuscitators relative to the absence of such factors indicates an effect by the PGHs and/or resuscitators according to the invention.
  • EXAMPLE 16 Using PGHs to Stimulate the Production of Isoflavanoids by the Fungus Glomus inter adices.
  • Isoflavanoids are commercially and medically important biological products naturally produced by plants and some fungi. Isoflavanoid compounds may be used as the synthetic starting point for the production of commercially useful compounds, or may be useful in their own right as, for example, immunosuppressants, antibiotics, antioxidants, or as drugs to treat or prevent diseases ranging in severity from baldness to cancer.
  • Isoflavanoids can be produced by the fungus Glomus interadices in a defined medium consisting of: (amounts are in mg/liter) KN0 3 610
  • Glomus interadices may be cultured in the above defined medium at temperatures ranging from 35-43°C and at an optimal pH of 7 (pH of about 6 to about 8 is acceptable). Isoflavanoids produced by the fungus may be quantitated by nuclear magnetic resonance or by mass spectroscopy. To enhance the production of isoflavanoids by Glomus interadices, the fungus is inoculated into the defined medium in the presence of one or more PGHs either with, or without resuscitators.
  • Titrations as described herein may be used to determine the optimal concentrations (final concentrations from 0.001 to 10 mmol will generally be effective) and combination (IAA plus GAs, or GAs plus zeatin, for example) of PGHs and/or resuscitators effective to enhance the production of isoflavanoids by this fungus.
  • Enhanced production of isoflavanoids according to the invention is determined by comparing the amount of isoflavanoids produced in the presence of PGHs with the amount produced in the absence of PGHs.

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Abstract

L'invention concerne un procédé servant à accroître le taux métabolique de bactéries ou champignons, afin d'obtenir un produit déterminé, ce procédé comprenant l'étape consistant à faire croître les bactéries ou champignons en présence d'une hormone de croissance végétale en dose suffisante pour permettre un accroissement du taux métabolique de ces bactéries ou champignons et obtenir un produit déterminé par rapport à la production d'un produit déterminé par des bactéries ou champignons que l'on a fait croître en l'absence de ladite hormone de croissance végétale. L'invention concerne également un procédé d'accroissement du taux de croissance des bactéries ou champignons, lequel procédé comprend l'étape consistant à faire croître les bactéries ou champignons en présence d'une hormone de croissance végétale choisie dans le groupe constitué par des gibbérellines, des cytokinines, des acides abscissiques et des éthylènes, en dose suffisante pour permettre un accroissement du taux de croissance de ces bactéries ou champignons par rapport au taux de croissance de ceux-ci en l'absence de ladite hormone de croissance.
PCT/US1999/020491 1998-09-08 1999-09-07 Modulation du metabolisme microbien et/ou du taux de croissance au moyen d'hormones de croissance vegetales WO2000014268A1 (fr)

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