WO2001068803A2 - Enzymes, voies et organismes pour la fabrication d'un monomere polymerisable par un bioprocessus cellulaire integral - Google Patents

Enzymes, voies et organismes pour la fabrication d'un monomere polymerisable par un bioprocessus cellulaire integral Download PDF

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WO2001068803A2
WO2001068803A2 PCT/US2001/006941 US0106941W WO0168803A2 WO 2001068803 A2 WO2001068803 A2 WO 2001068803A2 US 0106941 W US0106941 W US 0106941W WO 0168803 A2 WO0168803 A2 WO 0168803A2
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coa
dna sequence
itaconyl
hydroxybutyrate
itaconate
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WO2001068803A3 (fr
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Sergey A. Selifonov
Gjalt Huisman
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Maxygen, Inc.
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Publication of WO2001068803A3 publication Critical patent/WO2001068803A3/fr

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • C12P7/62—Carboxylic acid esters

Definitions

  • Functionalized hydroxyacids, and their ester and salt equivalents are important building blocks in the production of various polyester polymers. While ample art exists on the biocatalytic or fermentative production of 3-hydroxyalkanoates and 4- hydroxyalkanoates, there are very few convenient methods, either biological or chemical, for making derivatives of these compounds with additional functional groups in an economically viable manner. Some additional functional groups, such as olefinic moieties, are especially important because they can be further used for various chemical elaborations and modifications of the polymer without interfering with preparation of the polymer backbone. This makes it possible to alter the properties of polymer compositions and blends in desirable ways.
  • MBL 2-methylene- ⁇ -butyrolactone
  • MBL is a monomer that is functionally compatible with the polyester backbone of 3- and 4-hydroxyalkanoates, and other hydroxy-acids, including 1 hydroxybutyrates.
  • the methylenic group in MBL is highly reactive (more reactive than methacrylate) and thus amenable to a variety of chemical reactions, including radical polymerizations akin to those used for preparation of poly(meth)acrylate compounds.
  • MBL Petroleum-based chemical syntheses of MBL have been described; however, they are not economical for large volume polymer manufacturing needs.
  • the occurrence of MBL has been reported in several organisms, including plants of the Lilaceae family (e.g., Peruvian lily, tulips, alstroemeria, hydrangea), as well as in a variety of other species (Laurus nobilis, Erythronium grandiflorum).
  • MBL is present in these sources only at low levels which are not cost effective to recover.
  • a biological process that would allow for a fermentative manufacturing of MBL from inexpensive, renewable carbon sources (e.g., simple sugars, carbohydrates, etc.) would be of significant utility and economic importance.
  • the present invention presents methods, artificially evolved enzymes, novel biochemical pathways and whole cell bioprocesses which are used to produce MBL, and polyhydroxyalkanoates containing MBL, in an efficient and cost- effective manner.
  • FIGURE 1 is a schematic illustration of an artificial regioselective reduction pathway for the enzymatic conversion of itaconate to 2-mefhylene-4( ⁇ )-hydroxybutyrate.
  • Figure 2 depicts the biosynthesis of 2-methylene-4( ⁇ )-hydroxybutyrate via an isopentenylpyrophosphate intermediate.
  • Figure 3 illustrates the production of a mevalonate substrate used in the biosynthesis of 2- methylene-4( ⁇ )-hydroxybutyrate from simple carbon sources.
  • PHAs Polyhydroxyalkanoates
  • the present invention provides methods for producing enzymes, biochemical pathways, and whole cell bioprocesses which convert readily available simple carbon sources into polymerizable substrates and ultimately into PHAs. Novel pathways for converting itaconate to the polymerizable substrates 2- methylene- ⁇ -butyrolactone (2MBL) and 3-methylene- ⁇ -butyrolactone (3MBL) are described. Novel enzymes for converting polymerizable substrates to PHAs are also provided. In one aspect, enzymatic conversions of simple carbon sources, key intermediates, and polymerizable substrates of PHAs are provided.
  • these enzymatic conversions are carried out by recombinant or isolated enzymes that are itaconate-itaconyl-CoA converting enzymes, succinate semialdehyde dehydrogenases, 4- hydroxybutyrate dehydrogenases, and polyhydroxyalkanoate polymerases.
  • the recombinant or isolated enzymes are artificially evolved enzymes.
  • One aspect of the invention relates to a novel biochemical pathway for converting itaconate to 2MBL via the intermediates of itaconyl-CoA, itaconic 4- semialdehyde and 2-methylene-4( ⁇ )-hydroxybutyrate.
  • Embodiments provide independent conversion steps which can be practiced in vitro or by transforming one or more suitable hosts, either singly or in combination.
  • microorganisms which accumulate itaconate are used as hosts.
  • a carbon source e.g., a sugar, a starch, a fatty acid or an alcohol is provided to the recombinant host which converts the carbon source to itaconate.
  • this conversion is carried out by a cis-aconitate decarboxylase encoded by an exogenous DNA.
  • Alternative embodiments provide a biochemical pathway for the enzymatic conversions of a carbon source to mevalonate, mevalonate to isopentyl diphosphate or isoprenyl diphophate, and isopentyl diphosphate or isoprenyl diphosphate to itaconate.
  • one or more of these conversions is carried out by an artificially evolved enzyme.
  • bulk fermentation processes are provided in which one or several recombinant microorganisms catalyzing individual or combinations of steps in the biosynthetic pathway are cultured and their products recovered.
  • the microorganisms producing 2- or 3-methylene-4( ⁇ )-hydroxybutyrate are grown in an aqueous medium which is acidified to convert 2- or 3- methylene-4( ⁇ )-hydroxybutyrate to 2-or 3-methylene butyrolactone (MBL).
  • MBL 2-or 3-methylene butyrolactone
  • plants transfected with polynucleotides encoding the catalytic enzymes of the invention produce one or more of the substrates or products.
  • one or more conversion step is performed by an artificially evolved enzyme, for example by an artificially evolved enzyme derived, e.g., by DNA shuffling and/or other diversity generating procedures, from one or more polynucleotides encoding enzymes, or fragments thereof, of a bacterium or other organism.
  • the artificially evolved enzymes of the invention are improved relative to the parental enzyme, for at least one enzymatic activity, e.g., conversion rate, substrate specificity or the like.
  • an enzyme with itaconate-itaconyl-CoA converting activity is artificially evolved from a sucC and/or sucD gene derived from E.coli, H. influenza, R. prowazekii, B. subt ⁇ lis, M. tuberculae, C. trachomatis, C. pneumoniae, A. fulgidus, P. aeruginosa, T. aquaticus, C. kluyveri or A. aeolicus, or from a Pseudomonas species.
  • the itaconate-itaconyl-CoA converting enzyme is selected from among an acyl-CoA synthetase, a succinyl-CoA synthetase, an acetyl-CoA transferase, a succinyl-CoA transferase, a citrate-CoA transferase, a glutaconate-CoA transf erase and a 4-hydroxybutyrate-CoA transferase.
  • an enzyme that converts itaconyl-CoA to itaconic 4- semialdehyde is artificially evolved to increase conversion rate or alter substrate specificity of a parental enzyme.
  • the parental enzyme is encoded by a DNA sequence from e.g., C. kluyveri, R. eutropha or E. coli.
  • itaconic-4-semialdehyde is enzymatically converted to 2-methylene-4( ⁇ )-hydroxybutyrate.
  • the enzyme is artificially evolved to have a higher conversion rate, or an altered substrate specificity relative to a parental enzyme.
  • the artificially evolved enzyme is derived from a C. kluyveri 4-hydroxybutyrate dehydrogenase.
  • Another aspect of the invention provides for the production of polyhydroxyalkanoates from itaconate, 2-methylene-4( ⁇ )-hydroxybutyrate or 3-methylene- 4( ⁇ )-hydroxybutyrate by a recombinant or isolated polyhydroxyalkanoate polymerase.
  • the polyhydroxyalkanoate polymerase is an artificially evolved enzyme.
  • the polyhydroxyalkanoate polymerase is derived by DNA shuffling from aphaC gene of Aeromonas caviae.
  • the polymerization of the polyhydroxyalkanoate occurs in vitro.
  • the polymerization occurs in a recombinant cell, such as a bacterium, a plant cell or a fungal cell.
  • Methods for producing the artificially evolved enzymes e.g., by DNA shuffling are provided.
  • DNA fragments encoding an enzyme, or a sub-portion thereof, in a biochemical pathway for converting a carbon source to a polyhydroxyalkanoate are, for example, recursively recombined.
  • the resulting recombinant DNA segments are then screened for desirable enzymatic activities.
  • DNA segments that encode enzymes with improved conversion rates or substrate specificities are selected.
  • SucC, SucD and phaC genes are selected in some embodiments.
  • an “enzyme” is a biological catalyst, for the purpose of this disclosure, enzymes that are proteins, or polypeptides, will be considered.
  • the "enzymatic activity" of a protein refers to its activity catalyzing a chemical or biochemical reaction resulting in the conversion of a substrate to a product.
  • an enzyme is defined by its activity.
  • Corresponding nucleic acids are defined by the activity of an encoded protein or polypeptide, or by the activity encoded by a substantially similar nucleic acid sequence.
  • an enzyme that converts itaconate to itaconyl-CoA is referred to alternatively as a "itaconate-itaconyl-CoA converting enzyme, an acyl-CoA synthetase, a succinyl-CoA synthetase, an acetyl-CoA transferase, a succinyl-CoA transferase, a citrate-CoA transferase, a glutaconate-CoA transferase, or a 4-hydroxybutyrate-CoA transferase.
  • An enzyme that converts itaconyl-CoA to itaconic semialdehyde is referred to as an "itaconic semialdehyde dehydrogenase" or a succinate-semialdehyde dehydrogenase.
  • An enzyme that converts itaconic semialdehyde to 2-methylene-4( ⁇ )-hydroxybutyrate is referred to as a 4-hydroxybutyrate dehydrogenase.
  • An enzyme that polymerizes a polyhydroxyalkanoate is referred to alternatively as a polyhydroxyalkanoate (PHA) polymerase or a polyhydroxyalkanoate (PHA) synthase.
  • Enzymes of the invention can be "artificially evolved" enzymes, that is, they have been produced by one or a combination of methods for engineering desired changes into a nucleic acid, and thus, into the protein or polypeptide that the nucleic acid encodes. Examples of such methods include cloning, rational protein design strategies, cassette mutagenesis, and mutagenic recombination procedures, e.g., recursive recombination, such as, DNA shuffling techniques.
  • a wide variety of enzyme attributes are subject to the artificial evolution techniques of the present invention. These include: kinetic constants, stability, selectivity, inhibition profiles, altered substrate specificity, conversion rate, increased gene expression, activity under diverse environmental conditions (i.e., increased thermostability, increased activity in various organic solvents, pH tolerance, etc.), and the like.
  • conversion rate is a measure of an enzyme's activity and refers to the rate, e.g., molecules per unit time, at which substrate is converted to product.
  • substrate specificity refers to the biochemical molecule, or molecules that an enzyme recognizes and exerts a catalytic effect upon, often as compared to other substrate molecules.
  • Nucleic acids encoding the enzymes of the invention are also a feature of the invention. Such nucleic acids can be either DNA or RNA. The following attributes apply equally to a “nucleic acid” or to a “DNA” (or “RNA”).
  • An “exogenous” nucleic acid is a nucleic acid not produced by that cell type in nature.
  • a “parental” nucleic acid refers to a nucleic acid, optionally encoding a polypeptide, that serves as the starting material in an artificial evolution protocol, e.g., DNA shuffling.
  • the present invention provides alternative approaches for production and optimization of enzymes, biochemical pathways and recombinant organisms useful for producing methylene butyrolactone (MBL) by a whole cell bioprocess. Biosynthetic reactions leading to production of MBL, as well as the enzymes and genes involved have not been previously elucidated.
  • MBL is recognized as a metabolite of several alternative artificial metabolic pathways that are constructed using well- established molecular biology techniques, and artificially evolved, e.g., by DNA shuffling and other techniques, to optimize performance. Iterative recombination and selection procedures are particularly useful for the rapid improvement of the enzymes and pathways of the invention with respect to catalytic properties, (e.g., conversion rate, substrate specificity), product inhibition, level of expression, codon usage and the like.
  • catalytic properties e.g., conversion rate, substrate specificity
  • a fermentative pathway via enzymatic reduction of itaconyl-Coenzyme A 2) a fermentative pathway via biosynthesis of isopentenylpyrophosphate followed by a monooxygenase step applied to the allylic methyl group of the oxygenated hemiterpene intermediate; and 3) a biotransformation pathway from a methylsubstituted aromatic compound via a modified ort zo-fission pathway leading to itaconyl-CoA, further reduced as in the type (1) pathway.
  • MBL or its open ring form, methylene- 4( ⁇ )-hydroxybutyrate
  • Itaconic acid is a well known industrial product used in paper coatings and other applications. It is economically produced (approximate cost $2.00 U.SVlb, Chemical Market Reporter, 1999) by fermentation using a variety of fungal organisms utilizing a variety of carbohydrate sources. Levels of accumulated itaconic acid in fermentation as high as 75 g/L using Aspergillus terreus NRRL 1960 have been described.
  • bioprocesses employing reduction of a carboxyl group of itaconate involve microorganisms expressing an itaconate reduction pathway that are supplied with exogenous itaconic acid in the growth medium. Such processes can be either aerobic or anaerobic, and typically require an additional electron source.
  • the genes encoding enzymes for the regioselective reduction of itaconate intermediates can be expressed in microorganisms that produce endogenous itaconic acid, e.g., A. terreus NRRL 1960.
  • This approach provides for the direct conversion of an inexpensive carbon source (e.g., starch or glucose) to MBL or its open form (2- methylene-4( ⁇ )-hydroxybutyrate, 2-MHB) as the principal final fermentation product instead of itaconic acid.
  • a further advantage of this approach is that it results in the production of a less acidic monocarboxylic end-product (relative to the stronger diacid, itaconate) and allows the accumulation of a higher concentration of the desirable MBL monomer in the fermentation medium compared to that achievable for itaconate production.
  • Biological reduction of itaconic acid to MBL has not been described in the prior art. Prior to the present invention it was unobvious which genes and enzymes were required to accomplish such a process in a microorganism.
  • the invention provides an enumeration of the genes required for the regioselective reduction of itaconate to MBL or its open form, as well as methods for achieving significant improvements in the properties of these enzymes with respect to the production
  • any set of nucleic acids encoding enzymes, or fragments thereof e.g., synthesized oligonucleotides and/or nuclease digested nucleic acids
  • that perform a conversion in the pathway between itaconate and MBL, or between a simple carbon source and itaconate or another intermediate, or that perform the polymerization of a polyhydroxyalkanoate from MBL can be utilized in the methods described in this disclosure to produce new enzymes, and pathways, having the properties noted throughout.
  • Exemplary sets of nucleic acids are indicated in Tables 1 and 2, along with the corresponding accession numbers.
  • the MBL lactone form is a particularly convenient form of fermentation end-product as its solubility in aqueous medium is limited and it can be readily recovered by organic phase separation, distillation or organic solvent extraction techniques. Conversion of itaconate to MBL or its free acid can, for example, be effectively achieved by recruiting the corresponding genes from the Clostridium kluyveri succinate metabolic pathway (Sohling and Gottschalk (1993) Eur J Biochem 212:121; Genbank locus CLOCAT1A, L21902). Numerous other genes from a wide variety of microorganisms are also suitable, including but not limited to the illustrative examples provided in Table 1.
  • genes can be recruited to effect formation of itaconyl-CoA from itaconate, and itaconic semialdehyde to 2-methylene-4( ⁇ )-hydroxybutyrate, (or alternatively to 3-methylene-4( ⁇ )-hydroxybutyrate via regioselective reduction of the C(l) carbon.
  • the key step of the itaconate reduction pathway is reduction of the itaconyl-CoA to itaconic semialdehyde.
  • the coenzymeA dependent succinate-semialdehyde dehydrogenase gene from Clostridium kluyveri (Sohling and Gottschalk, supra) is utilized.
  • homologous genes from other organisms can be isolated by methods known in the art and recruited for this purpose (see, e.g., Table 1).
  • Genes encoding enzymes in the pathway for converting itaconate to 2-MHB or 3-MHB are isolated by methods well-known in the art, e.g., cloning, amplification, transformation, etc. (see, discussion below), and expressed singly or in combination in microbial hosts, e.g., E. coli, to convert exogenously supplied itaconate to MBL.
  • microorganisms that produce itaconic acid e.g., the fungus Aspergillus terreus NRRL1960
  • fungus Aspergillus terreus NRRL1960 are used as the starting material for engineering MBL producing organisms.
  • Other fungal strains which have cis-aconitate decarboxylase activity can also be used as host strains; however, preparation of mutants that are deficient in itaconyl-CoA hydratase is required to increase levels of itaconate production.
  • Such fungal strains can be used to heterologously express the above enzymes for itaconate reduction via itaconyl-CoA and itaconic semialdehyde to 2-methylene-4( ⁇ )-hydroxybutyrate.
  • Another alternative is to heterologously express a cis-aconitate decarboxylase (E.C.4.1.1.6) from, e.g., A. terreus NRRL1960, or similar itaconate- accumulating organism, in a microorganism transformed with the itaconate reduction pathway described above.
  • a cis-aconitate decarboxylase E.C.4.1.1.6
  • Such genes can be isolated by one skilled in the art by well- established methods, or can be artificially evolved, e.g., by DNA shuffling as described below, using starting sequences selected from, but not limited to, those in Table 2.
  • Table 1 Sources of starting material for obtaining enzymes that convert itaconate to
  • the pathways described above for the production of itaconate, the conversion of itaconate to MBL, and optionally for the polymerization of polyhydroxyalkanoates can be expressed in a plant species, (e.g., an oil producing species such as rape, canola or sunflower, among many others).
  • a plant species e.g., an oil producing species such as rape, canola or sunflower, among many others.
  • MBL (or its free acid forms, 2-methylene-4( ⁇ )-hydroxybutyrate and 3- methylene-4( ⁇ )-hydroxybutyrate) is also recognized in the present invention as a hemi- terpenoid (isoprene derivative) compound with two oxygen-containing functions: a carboxyl at the "tail” and a hydroxyl at the "head.” Therefore, alternative biosynthetic pathways for the biosynthesis of MBL in vivo are provided departing from the well-known intermediates of terpenoid biosynthesis, isopentenyl diphosphate (pyrophosphate).
  • pyrophosphate isopentenyl diphosphate
  • Isopentenyl diphosphate is, in turn, provided by a mevalonate- dependent or independent biosynthetic pathway.
  • a mevalonate- dependent pathway as illustrated in Figure 3, is utilized, as all the genes of the mevalonate- dependent pathway are well known in the art, are available from multiple source organisms, and can be heterologously expressed in a variety of microbial hosts, including bacteria and yeast.
  • Preferred organisms are mutated or selected to have a low activity of isopentenyl- diphosphate isomerase (e.g., conversion of isopentenyl diphosphate to dimethylallyl diphosphate), and reduced activity of isopropenyl/dimethylallyl condensing enzymes involved in the formation of isoprenoid intermediates with C 10 or longer.
  • Isopentenyl diphosphate provides the correct carbon backbone for MBL biosynthesis. However, it requires oxidation at the allylic methyl group.
  • aromatic biodegradation pathways involving intermediate formation of 3-methylmuconic acid from 4-methylcatechol can be used in conjunction with the co-expression of enzymes or genes allowing for the subsequent reactions leading to the formation of itaconyl-CoA.
  • Trichosporon cutaneum fungus can be used as a source of the genes encoding enzymes of the modified beta-ketoadipate pathway to itaconyl-CoA.
  • genes can be isolated by methods well-known in the art, and heterologously expressed in a suitable host organism. Indeed, only genes for enzymes catalyzing the steps subsequent to methylmuconolactone formation need be isolated, as the genes converting 4-methylcatechol to 3-methylmuconate and methylmuconolactone can be easily recruited from a variety of bacterial sources.
  • the organism used for MBL production is deficient in converting activity of catechol-2,3- dioxygenase or any other meta-fission dioxygenase capable of cleaving 4-methylcatechol at positions 2,3 and/or 1,6.
  • This can be readily achieved by selecting appropriate host strains, and/ or by disabling meta-fission activity by means of mutagenesis or gene deletion. It is also possible to recruit ortho-fission, e.g., 1,2-dioxygenase genes from alternative sources known in the art, sequences of which are available from Genbank.
  • PHAs Polyhydroxyalkanoates
  • the phaC gene of Aeromonas caviae is shuffled by any of the described techniques and the resulting nucleic acids are transfected into a suitable microorganism for screening, e.g., E. coli.
  • the transformed microorganisms expressing the shuffled nucleic acids are grown in the presence of itaconate, 2-MHB, or 3-MHB, and evaluated in the presence of Nile Red.
  • Clones expressing an active PHA polymerase and which form PHA appear white and fluoresce in the presence of Nile Red, while negative colonies appear gray and do not fluoresce.
  • a whole genome shuffling approach utilizing A. caviae, as described below, can be employed.
  • the foregoing has described novel biosynthetic pathways for the conversion of itaconate to MBL or its open ring form.
  • the present invention provides methods for artificially evolving these enzymes, pathways and organisms, to optimize the production of the desired product, or intermediates. This is achieved by various diversity generating and selection procedures known in the art, including, e.g., DNA shuffling, as described by the authors and their coworkers.
  • a variety of diversity generating procedures are available and described in the art.
  • the procedures can be used separately, and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well variants of encoded proteins.
  • Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and or organisms with new and/or improved characteristics.
  • any of the diversity generating procedures described herein can be the generation of one or more nucleic acids, which can be selected or screened for nucleic acids that encode proteins with or which confer desirable properties.
  • any nucleic acids that are produced can be selected for a desired activity or property, e.g. increased catalytic activity, altered substrate specificity.
  • a variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner. Descriptions of a variety of diversity generating procedures suitable for producing modified nucleic acid sequences encoding enzymes contributing to biosynthetic pathways for the conversion of itaconate to MBL, and for the polymerization of polyhydroxyalkanoates, are found in the following publications and the references cited therein: Soong, N. et al. (2000) "Molecular breeding of viruses” Nat Genet 25(4):436-439; Stemmer, et al.
  • Mutational methods of generating diversity include, for example, site- directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al. (1996) “Oligonucleotide-directed random mutagenesis using the phosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis” Science 229:1193-1201; Carter (1986) "Site-directed mutagenesis” Biochem. J.
  • sequence modification methods such as mutation, recombination, etc.
  • any of the methods described above can be adapted to the present invention to evolve enzymes, novel biochemical pathways and whole cell bioprocesses, and/or optimize the same, for the production of polyhydroxyalkanoates
  • Nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
  • DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
  • sexual PCR mutagenesis can be used in which random (or pseudo random, or even non- random) fragmentation of the DNA molecule is followed by recombination, based on sequence similarity, between DNA molecules with different but related DNA sequences, in vitro, followed by fixation of the crossover by extension in a polymerase chain reaction.
  • genes encoding the enzymes for the production of MBL or polyhydroxyalkanoates of the invention can be recombined in vitro, e.g., by DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
  • nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells.
  • Many such in vivo recombination formats are set forth in the references noted above. Such formats optionally provide direct recombination between nucleic acids of interest, or provide recombination between vectors, viruses, plasmids, etc., comprising the nucleic acids of interest, as well as other formats. Details regarding such procedures are found in the references noted above.
  • nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between exogenous and or endogenous sequences encoding the enzymes of the invention, e.g., plasmids, nucleic acids in cells.
  • Whole genome recombination methods can also be used in which whole genomes of cells or other organisms are recombined, optionally including spiking of the genomic recombination mixtures with desired library components (e.g., genes encoding enzymes of the pathways for synthesizing MBL and/or polyhydroxyalkanoates of the present invention). These methods have many applications, including those in which the identity of a target gene is not known. Details on such methods are found, e.g., in WO 98/31837 by del Cardayre et al.
  • Synthetic recombination methods can also be used, in which oligonucleotides corresponding to targets of interest are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which correspond to more than one parental nucleic acid, thereby generating new recombined nucleic acids.
  • Oligonucleotides can be made by standard nucleotide addition methods, or can be made, e.g., by tri-nucleotide synthetic approaches.
  • the resulting recombined sequence strings are optionally converted into nucleic acids by synthesis of nucleic acids which correspond to the recombined sequences, e.g., in concert with oligonucleotide synthesis/ gene reassembly techniques. This approach can generate random, partially random or designed variants.
  • the parental polynucleotide strand can be removed by digestion (e.g., if RNA or uracil-containing), magnetic separation under denaturing conditions (if labeled in a manner conducive to such separation) and other available separation/purification methods.
  • the parental strand is optionally co-purified with the chimeric strands and removed during subsequent screening and processing steps. Additional details regarding this approach are found, e.g., in "SINGLE-STRANDED
  • NUCLEIC ACID TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION by Affholter, USSN 09/656,549, filed Sept. 6, 2000.
  • single-stranded molecules are converted to double- stranded DNA (dsDNA) and the dsDNA molecules are bound to a solid support by ligand- mediated binding. After separation of unbound DNA, the selected DNA molecules are released from the support and introduced into a suitable host cell to generate a library enriched sequences which hybridize to the probe.
  • dsDNA double- stranded DNA
  • a library produced in this manner provides a desirable substrate for further diversification using any of the procedures described herein.
  • any of the preceding general recombination formats can be practiced in a reiterative fashion (e.g., one or more cycles of mutation/recombination or other diversity generation methods, optionally followed by one or more selection methods) to generate a more diverse set of recombinant nucleic acids.
  • Mutagenesis employing polynucleotide chain termination methods have also been proposed (see e.g., U.S. Patent No. 5,965,408, "Method of DNA reassembly by interrupting synthesis” to Short, and the references above), and can be applied to the present invention.
  • double stranded DNAs corresponding to one or more genes sharing regions of sequence similarity are combined and denatured, in the presence or absence of primers specific for the gene.
  • the single stranded polynucleotides are then annealed and incubated in the presence of a polymerase and a chain terminating reagent (e.g., ultraviolet, gamma or X-ray irradiation; ethidium bromide or other intercalators; DNA binding proteins, such as single strand binding proteins, transcription activating factors, or histones; polycyclic aromatic hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated polymerization mediated by rapid thermocycling; and the like), resulting in the production of partial duplex molecules.
  • a chain terminating reagent e.g., ultraviolet, gamma or X-ray irradiation; ethidium bromide or other intercalators; DNA binding proteins, such as single strand binding proteins, transcription activating factors, or histones; polycyclic aromatic hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated poly
  • the partial duplex molecules e.g., containing partially extended chains, are then denatured and reannealed in subsequent rounds of replication or partial replication resulting in polynucleotides which share varying degrees of sequence similarity and which are diversified with respect to the starting population of DNA molecules.
  • the products, or partial pools of the products can be amplified at one or more stages in the process.
  • Polynucleotides produced by a chain termination method, such as described above, are suitable substrates for any other described recombination format.
  • Mutational methods which result in the alteration of individual nucleotides or groups of contiguous or non-contiguous nucleotides can be favorably employed to introduce nucleotide diversity into the nucleic acids encoding the enzymes of the invention.
  • Many mutagenesis methods are found in the above-cited references; additional details regarding mutagenesis methods can be found in following, which can also be applied to the present invention.
  • error-prone PCR can be used to generate nucleic acid variants.
  • PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Examples of such techniques are found in the references above and, e.g., in Leung et al. (1989) Technique 1:11-15 and Caldwell et al. (1992) PCR Methods Applic. 2:28-33.
  • assembly PCR can be used, in a process which involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions can occur in parallel in the same reaction mixture, with the products of one reaction priming the products of another reaction.
  • Oligonucleotide directed mutagenesis can be used to introduce site-specific mutations in a nucleic acid sequence of interest. Examples of such techniques are found in the references above and, e.g., in Reidhaar-Olson et al. (1988) Science, 241:53-57.
  • cassette mutagenesis can be used in a process that replaces a small region of a double stranded DNA molecule with a synthetic oligonucleotide cassette that differs from the native sequence.
  • the oligonucleotide can contain, e.g., completely and/or partially randomized native sequence(s).
  • Recursive ensemble mutagenesis is a process in which an algorithm for protein mutagenesis is used to produce diverse populations of phenotypically related mutants, members of which differ in amino acid sequence. This method uses a feedback mechanism to monitor successive rounds of combinatorial cassette mutagenesis. Examples of this approach are found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811- 7815.
  • Exponential ensemble mutagenesis can be used for generating combinatorial libraries with a high percentage of unique and functional mutants. Small groups of residues in a sequence of interest are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Examples of such procedures are found in Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552.
  • In vivo mutagenesis can be used to generate random mutations in any cloned DNA of interest by propagating the DNA, e.g., in a strain of E. coli that carries mutations in one or more of the DNA repair pathways. These "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA. Such procedures are described in the references noted above.
  • Transformation of a suitable host with such multimers consisting of genes that are divergent with respect to one another, (e.g., derived from natural diversity or through application of site directed mutagenesis, error prone PCR, passage through mutagenic bacterial strains, and the like), provides a source of nucleic acid diversity for DNA diversification, e.g., by an in vivo recombination process as indicated above.
  • a multiplicity of monomeric polynucleotides sharing regions of partial sequence similarity can be transformed into a host species and recombined in vivo by the host cell. Subsequent rounds of cell division can be used to generate libraries, members of which, include a single, homogenous population, or pool of monomeric polynucleotides.
  • the monomeric nucleic acid can be recovered by standard techniques, e.g., PCR and/or cloning, and recombined in any of the recombination formats, including recursive recombination formats, described above.
  • Multispecies expression libraries include, in general, libraries comprising cDNA or genomic sequences from a plurality of species or strains, operably linked to appropriate regulatory sequences, in an expression cassette.
  • the cDNA and/or genomic sequences are optionally randomly ligated to further enhance diversity.
  • the vector can be a shuttle vector suitable for transformation and expression in more than one species of host organism, e.g., bacterial species, eukaryotic cells.
  • the library is biased by preselecting sequences which encode a protein of interest, or which hybridize to a nucleic acid of interest. Any such libraries can be provided as substrates for any of the methods herein described.
  • recombined CDRs derived from B cell cDNA libraries can be amplified and assembled into framework regions (e.g., Jirholt et al. (1998) "Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework” Gene 215: 471) prior to diversifying according to any of the methods described herein. Libraries can be biased towards nucleic acids which encode proteins with desirable enzyme activities.
  • the clone can be mutagenized using any known method for introducing DNA alterations.
  • a library comprising the mutagenized homologues is then screened for a desired activity, which can be the same as or different from the initially specified activity.
  • Desired activities can be identified by any method known in the art.
  • WO 99/10539 proposes that gene libraries can be screened by combining extracts from the gene library with components obtained from metabolically rich cells and identifying combinations which exhibit the desired activity.
  • clones with desired activities can be identified by inserting bioactive substrates into samples of the library, and detecting bioactive fluorescence corresponding to the product of a desired activity using a fluorescent analyzer, e.g., a flow cytometry device, a CCD, a fluorometer, or a spectrophotometer.
  • a fluorescent analyzer e.g., a flow cytometry device, a CCD, a fluorometer, or a spectrophotometer.
  • Libraries can also be biased towards nucleic acids which have specified characteristics, e.g., hybridization to a selected nucleic acid probe.
  • a desired activity e.g., an enzymatic activity, for example: a lipase, an esterase, a protease, a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, a hydratase, a nitrilase, a transaminase, an amidase or an acylase) can be identified from among genomic DNA sequences in the following manner.
  • an enzymatic activity for example: a lipase, an esterase, a protease, a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an oxygenase, a peroxidase
  • Single stranded DNA molecules from a population of genomic DNA are hybridized to a ligand-conjugated probe.
  • the genomic DNA can be derived from either a cultivated or uncultivated microorganism, or from an environmental sample. Alternatively, the genomic DNA can be derived from a multicellular organism, or a tissue derived therefrom.
  • Second strand synthesis can be conducted directly from the hybridization probe used in the capture, with or without prior release from the capture medium or by a wide variety of other strategies known in the art.
  • the isolated single-stranded genomic DNA population can be fragmented without further cloning and used directly in, e.g., a recombination-based approach, that employs a single-stranded template, as described above.
  • Non-Stochastic methods of generating nucleic acids and polypeptides are alleged in Short “Non-Stochastic Generation of Genetic Vaccines and Enzymes” WO 00/46344. These methods, including proposed non-stochastic polynucleotide reassembly and site-saturation mutagenesis methods be applied to the present invention as well.
  • Random or semi-random mutagenesis using doped or degenerate oligonucleotides is also described in, e.g., Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10:297-300; Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods Enzymol. 208:564-86; Lim and Sauer (1991) "The role of internal packing interactions in determining the structure and stability of a protein” J. Mol. Biol.
  • kits for mutagenesis, library construction and other diversity generation methods are also commercially available.
  • kits are available from, e.g., Stratagene (e.g., QuickChangeTM site-directed mutagenesis kit; and ChameleonTM double- stranded, site-directed mutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkel method described above), Boehringer Mannheim Corp., Clonetech Laboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3 prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs, Pharmacia Biotech, Promega Corp., Quantum Biotechnologies, Amersham International pic (e.g., using the Eckstein method above), and Boothn Biotechnology Ltd (e.g., using the Carter/Winter method above).
  • Stratagene e.g., QuickChangeTM site-directed mutagenesis kit
  • nucleic acids of the invention can be recombined (with each other, or with related (or even unrelated) sequences) to produce a diverse set of recombinant nucleic acids, including, e.g., sets of homologous nucleic acids, as well as corresponding polypeptides.
  • the above methodologies either singly or in combination are used to evolve and optimize enzymes and pathways which convert itaconate to MBL and/or polymerize MBL to polyhydroxyalkanoates.
  • the methods of the invention are adapted to each application through the choice of substrates and the methods of screening or selection.
  • enzymes with increased conversion rates or altered substrate specificity can be selected by monitoring production of a specified product or intermediate, by any technique known in the art
  • in vitro techniques based on the characteristics of polynucleotides, such as PCR, LCR, nucleic acid hybridization analysis, or on the characteristics of proteins, e.g. western hybridization, proteomics, are the method of choice in some instances.
  • biocatalysts of the present invention can be evolved, including assorted kinetic constants, stability, selectivity, inhibition profiles, altered substrate specificity, increased activity, increased gene expression, activity under diverse environmental conditions (i.e., increased thermostability, increased activity in various organic solvents, pH tolerance, etc.), and the like.
  • one or more recombination and/or mutagenesis cycle(s) is/are optionally followed by at least one cycle of selection for molecules having one or more of these or other desired traits or properties.
  • a recombination or mutagenesis procedure is performed in vitro, the products of recombination, i.e., recombinant or shuffled nucleic acids, are sometimes introduced into cells before the selection step.
  • Recombinant nucleic acids can also be linked to an appropriate vector or to other regulatory sequences before selection.
  • products of recombination and/or mutagenesis generated in vitro are sometimes packaged in viruses (e.g., bacteriophage) before selection.
  • viruses e.g., bacteriophage
  • recombinant segments are extracted from the cells, and optionally packaged as viruses or other vectors, before selection.
  • the nature of selection depends on what trait or property is to be acquired or for which improvement is sought. It is not usually necessary to understand the molecular basis by which particular recombination products have acquired new or improved traits or properties relative to the starting substrates.
  • a gene has many component sequences, each having a different intended role (e.g., coding sequences, regulatory sequences, targeting sequences, stability-conferring sequences, subunit sequences and sequences affecting integration). Each of these component sequences are optionally varied, e.g., recombined, simultaneously. Selection is then performed, for example, for recombinant products that have an increased ability to confer activity upon a cell without the need to attribute such improvement to any of the individual component sequences of the vector.
  • initial round(s) of screening can sometimes be performed using bacterial cells due to high transfection efficiencies and ease of culture.
  • yeast, fungal or other eukaryotic systems may also be used for library expression and screening when bacterial expression is not practical or desired.
  • other types of selection that are not amenable to screening in bacterial or simple eukaryotic library cells, are performed in cells selected for use in an environment close to that of their intended use. Final rounds of screening are optionally performed in the precise cell type of intended use.
  • At least one and usually a collection of recombinant products surviving a first round of screening/selection are optionally subject to a further round of diversification, e.g., recombination and/or mutagenesis.
  • These recombinant products can be, e.g., recombined with each other or with exogenous segments representing the original substrates or further variants thereof.
  • the process e.g., recombination, can proceed in vitro or in vivo.
  • the components can be subjected to further recombination in vivo, or can be subjected to further recombination in vitro, or can be isolated before performing a round of in vitro recombination.
  • the previous selection step identifies desired recombinant products in naked form or as components of viruses, these segments can be introduced into cells to perform a round of in vivo recombination.
  • the second round of recombination irrespective how performed, generates additionally recombined products which encompass more diversity than is present in recombinant products resulting from previous rounds.
  • the second round of recombination may be followed by still further rounds of screening/selection according to the principles discussed for the first round.
  • the stringency of selection can be increased between rounds.
  • the nature of the screen and the trait or property being selected may be varied between rounds if improvement in more than one trait or property is sought. Additional rounds of recombination and screening can then be performed until the recombinant products have sufficiently evolved to acquire the desired new or improved trait or property.
  • Itaconate is a strong dicarboxylic acid
  • the latter two compounds of interest can be extracted selectively into organic solvent at a pH between the second pK of itaconate and the pK of 2-methylene-4( ⁇ )-hydroxybutyrate.
  • This can be established readily empirically for each assay condition, and reproducibly attained by adding defined amounts of strong mineral acids, e.g., sulfuric, hydrochloric, nitric, etc., or a strong inorganic buffer with pH in the above specified interval.
  • the solvent for extraction is preferably selected from solvents which are lighter than water, have moderate or low volatility, and are transparent to ultraviolet (UV) light, in the range that allows for UV measurements of the acrylic methylenic double bond of MBL.
  • MBL detection makes use of the chemical reactivity of the methylenic double bond of MBL.
  • oxidizing reagents e.g., permanganate, halogen solutions
  • thiol reagents can be used in conjunction with various chromophoric co-reagents to measure amounts of extractable olefins, and thus to assess MBL concentrations.
  • nucleic acids encoding the enzymes of the invention can be prepared using various methods or combinations thereof, including certain DNA synthetic techniques (e.g., mononucleotide- and/or trinucleotide-based synthesis, reverse- transcription, etc.), DNA amplification, nuclease digestion, etc.
  • DNA synthetic techniques e.g., mononucleotide- and/or trinucleotide-based synthesis, reverse- transcription, etc.
  • DNA amplification e.g., DNA amplification
  • nuclease digestion e.g., DNA amplification, DNA ase digestion, etc.
  • the identification and acquisition of desirable substrate nucleic acids can be facilitated by a variety of means. For example, selection algorithms can be used to identify sequences in public or proprietary databases which meet any user-selected criterion for substrate selection. These user criteria include, activity, encoded activity, homology, public availability, and any other criteria of interest.
  • character strings corresponding to nucleic acids can be generated according to any set of criteria selected by the user, including similarity to existing sequences, modification of an existing sequence according to any desired modification parameter (genetic algorithm, etc.), random, or non-random (e.g., weighted) sequence generation, etc.
  • Data structures comprising diverse sequences can be formed in a digital or analog computer or in a computer readable medium and the data structures converted from character strings to nucleic acids for subsequent physical manipulations.
  • Either computer data or nucleic acids can be "data structures," a term which refers to the organization and optionally associated device for the storage of information, typically comprising multiple "pieces" of information.
  • the data structure can be a simple recordation of the information (e.g., a list) or the data structure can contain additional information (e.g., annotations) regarding the information contained therein, can establish relationships between the various "members" (information "pieces") of the data structure, and can provide pointers or linked to resources external to the data structure.
  • the data structure can be intangible but is rendered tangible when stored/represented in tangible medium.
  • the data structure can represent various information architectures including, but not limited to simple lists, linked lists, indexed lists, data tables, indexes, hash indices, flat file databases, relational databases, local databases, distributed database, thin client databases, and the like.
  • Nucleic acids can be selected by the user based upon sequence similarity to one or more additional nucleic acids.
  • BLAST Basic Local Alignment Search Tool
  • BLAST Basic Local Alignment Search Tool
  • nucleic acid databases can be utilized during the nucleic acid sequence selection and/or design processes.
  • Genbank®, Entrez®, EMBL, DDBJ, GSDB, NDB and the NCBI are examples of public database/search services that can be accessed. These databases are generally available via the internet or on a contract basis from a variety of companies specializing in genomic information generation and/or storage. These and other helpful resources are readily available and known to those of skill.
  • sequence of a polynucleotide to be used in any of the methods of the present invention can also be readily determined using techniques well-known to those of skill, including Maxam-Gilbert, S anger Dideoxy, and Sequencing by Hybridization methods. For general descriptions of these processes consult, e.g., Stryer (1995) Biochemistry (4 th Ed.) W.H. Freeman and Company, New York, (“Stryer”) and Lewin (1997) Genes VI Oxford University Press, Oxford (“Lewin”). See also, Maxam and Gilbert (1977) Proc Natl Acad Sci USA 74:560, Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463, Hunkapiller et al.
  • sequence information can be used to design and synthesize target nucleic acid sequences corresponding to, e.g., overlapping nucleic acids encoding enzymes that perform a conversion step in the production of MBL, or polyhydroxyalkanoates from itaconate or other carbon source, or other nucleic acid fragments (e.g., for the oligonucleotide and in silico shuffling approaches noted above).
  • nucleic acid sequences are synthesized by the sequential addition of activated monomers and/or trimers to an elongating polynucleotide chain. See e.g., Caruthers, M.H. et al. (1992) Meth Enzymol 211:3. Additional details are supplied in U.S Patent application number 09/408,393 file USE OF CODON- VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING, herein incorporated by reference.
  • nucleic acid can optionally be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (www.genco.com), ExpressGen, Inc. (www.expressgen.com), Operon Technologies, Inc. (www.operon.com), and many others.
  • Nucleic acids encoding any of the enzymes described above can be derived from expression products, e.g., mRNAs expressed from genes within a cell of a plant or other organism. A number of techniques are available for detecting RNAs.
  • RNA can be converted into a double stranded DNA using a reverse transcriptase enzyme and a polymerase. See, Ausubel, Sambrook and Berger. Messenger RNAs can be detected by converting, e.g., mRNAs into cDNAs, which are subsequently detected in, e.g., a standard "Southern blot" format.
  • RNA polymerase mediated techniques e.g., NASBA.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA RNA polymerase mediated techniques
  • assembled sequences are checked, e.g., for incorporation of enzyme encoding nucleic acid subsequences. This can be done by cloning and sequencing the nucleic acids, and or by restriction digestion, e.g., as essentially taught in Ausubel, Sambrook, and Berger, supra.
  • sequences can be PCR amplified and sequenced directly.
  • additional PCR sequencing methodologies are also particularly useful.
  • nucleic acid sequences encoding one or more naturally occuring, mutant or artificially evolved enzyme are introduced into the cells of particular organisms of interest.
  • bacterial cells any of which may be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors, etc.
  • Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. Bacteria are typically grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook).
  • kits are commercially available for the purification of plasmids from bacteria. For their proper use, follow the manufacturer's instructions (see, for example, EasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanTM, from Stratagene; and, QIAexpress Expression SystemTM from Qiagen).
  • the isolated and purified plasmids are then further manipulated to produce other plasmids.
  • yeast cells can be transfected by preparation of spheroblasts or by treatment with alkaline salts.
  • DNA can be introduced into plant and fungal cells by, for example, electroporation, microinjection, PEG precipitation, or particle-mediated bombardement ("biolistics").
  • Agrobacterium mediated transformation can be used to introduce exogenous DNA sequences situated between T- DNA ends into plant protoplasts, plant tissue explants, and whole plants as well as fungal cells using appropriate strains and vectors.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman and Smith (1979) Gene 8:81; Roberts et al. (1987) Nature 328:731; Schneider et al.
  • the present invention provides computers, computer readable media and integrated systems comprising character strings corresponding nucleic acids encoding enzymes involved in the production of MBL. These sequences can be manipulated by in silico recombination, e.g., shuffling, methods, or by standard sequence alignment (also discussed, supra), or word processing software.
  • Integrated systems for analysis in the present invention typically include a digital computer with software for aligning or manipulating nucleic acid sequences as well as data sets entered into the software system comprising any of the sequences herein.
  • the computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, OS2TM WINDOWSTM WINDOWS NTTM, WINDOWS95TM, WINDOWS98TM LINUX based machine, a MACINTOSHTM, Power PC, or a UNIX based (e.g., SUNTM work station) machine) or other commercially common computer which is known to one of skill.
  • Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visual basic, Fortran, Basic, Java, or the like.
  • the computer system is used to perform in silico shuffling of character strings that correspond to nucleic acid fragments derived from the enzymes of the invention.
  • a variety of such methods are set forth in "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov and Stemmer, filed February 5, 1999 (USSN 60/118854) and "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov and Stemmer, filed October 12, 1999 (USSN 09/416,375).
  • genetic operators are used in genetic algorithms to change given sequences, for example sequences corresponding to nucleic acids that encode the enzymes of the invention, e.g., by mimicking genetic events such as mutation, recombination, death and the like.
  • Multi-dimensional analysis to optimize sequences can also be performed in the computer system, e.g., as described in the '375 application.
  • a digital system can also instruct an oligonucleotide synthesizer to synthesize oligonucleotides corresponding to one or more of the naturally occurring or altered enzymes used to synthesize a polyhydroxyalkanoate or a substrate thereof, e.g., used for gene reconstruction or recombination, or to order such oligonucleotides from commercial sources (e.g., by printing appropriate order forms or by linking to an order form on the internet).
  • the digital system can also include output elements for controlling nucleic acid synthesis, i.e., an integrated system of the invention optionally includes an oligonucleotide synthesizer or an oligonucleotide synthesis controller for synthesizing nucleic acid fragments.
  • the system can include other operations which occur downstream from an alignment or other operation performed using a character string corresponding to a sequence herein, e.g., as noted above with reference to assays.
  • the present invention also provides a kit or system (e.g., a bioreactor) for performing one or more of the enzyme catalyzed reactions described herein.
  • the kit or system can optionally include a set of instructions for practicing one or more of the methods described herein; one or more assay components that can include at least one recombinant, isolated and/or artificially evolved enzyme or at least one cell that includes one or more such enzymes or both, and one or more reagents; and a container for packaging the set of instructions and the assay components.
  • the assay component can optionally include at least one immobilized enzyme as described above, or at least one such enzyme free in solution, or both.
  • Recombinant, isolated, or artificially evolved enzymes, or a combination thereof can be supplied as assay components (e.g., bioreactors) of the kits or systems of the present invention, to catalyze the conversion of selected reactants to one or more products, e.g., the conversion of inexpensive carbon sources to useful products.
  • reactants can be added to a reaction mixture in solution, e.g., as a in batch method approach, or into the liquid stream of a continuous feed process.
  • the present invention provides for the use of any component or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.

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Abstract

L'invention concerne des procédés de production d'enzymes, des voies biochimiques et des bioprocessus cellulaire intégraux, permettant la conversion de sources de carbone simples en substrats polymérisables, et en polyhydroxyalcanoates (PHAs). Des voies artificielles pour la conversion d'itaconate en méthylène-η-butyrolactone de substrat polymérisable, par la réduction stéréosélective des carbones C(1) ou C(4) sont décrites. Des nouvelles enzymes pour la conversion desdits substrats polymérisables en PHH sont également décrites.
PCT/US2001/006941 2000-03-02 2001-02-28 Enzymes, voies et organismes pour la fabrication d'un monomere polymerisable par un bioprocessus cellulaire integral WO2001068803A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102205A1 (fr) * 2008-02-14 2009-08-20 Wageningen Universiteit Séquences nucléotidiques codant pour la cis-aconitique décarboxylase et son utilisation
WO2013033209A1 (fr) * 2011-08-29 2013-03-07 Novomer, Inc. Copolymères poly(hydroxy alkanoate)
CN105936887A (zh) * 2007-03-16 2016-09-14 基因组股份公司 用于1,4-丁二醇和其前体生物合成的组合物和方法
US10669537B2 (en) 2016-12-14 2020-06-02 Biological Research Centre Mutagenizing intracellular nucleic acids
EP2084209B1 (fr) * 2006-11-21 2021-06-02 LG Chem, Ltd. Copolymère comprenant un motif de 4-hydroxybutyrate et un motif de lactate et son procédé de fabrication
WO2023052538A1 (fr) * 2021-10-01 2023-04-06 Basf Se Voie biochimique pour la production de tulipaline a par l'intermédiaire d'acide itaconique
WO2023151894A1 (fr) 2022-02-11 2023-08-17 Henkel Ag & Co. Kgaa Méthode de synthèse d'alpha-méthylène-gamma-butyrolactone

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
COOPER ET AL.: 'The utilization of aconate and itaconate by micrococcus sp.' BIOCHEM. J. vol. 94, no. 1, 1965, pages 25 - 31, XP002905747 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2084209B1 (fr) * 2006-11-21 2021-06-02 LG Chem, Ltd. Copolymère comprenant un motif de 4-hydroxybutyrate et un motif de lactate et son procédé de fabrication
CN105936887A (zh) * 2007-03-16 2016-09-14 基因组股份公司 用于1,4-丁二醇和其前体生物合成的组合物和方法
WO2009102205A1 (fr) * 2008-02-14 2009-08-20 Wageningen Universiteit Séquences nucléotidiques codant pour la cis-aconitique décarboxylase et son utilisation
WO2013033209A1 (fr) * 2011-08-29 2013-03-07 Novomer, Inc. Copolymères poly(hydroxy alkanoate)
US10669537B2 (en) 2016-12-14 2020-06-02 Biological Research Centre Mutagenizing intracellular nucleic acids
WO2023052538A1 (fr) * 2021-10-01 2023-04-06 Basf Se Voie biochimique pour la production de tulipaline a par l'intermédiaire d'acide itaconique
WO2023151894A1 (fr) 2022-02-11 2023-08-17 Henkel Ag & Co. Kgaa Méthode de synthèse d'alpha-méthylène-gamma-butyrolactone

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