JP2003529328A - Evolution and use of enzymes for combinatorial and medicinal chemistry - Google Patents

Abstract

  (57) [Summary] The present invention provides a library of recombinant derivatizing enzymes useful for biocatalysis of derivatives of organic molecules (including lead compounds for pharmaceutical use). The recombinant derivatizing enzyme catalyzes a reaction such as modification or substitution of a functional group on an organic molecule or addition of a chemical moiety onto an existing functional group. The use of a recombinant enzyme library can provide an enzyme that catalyzes the formation of an organic molecule derivative that cannot be made using only naturally occurring enzymes.

Description

Detailed Description of the Invention

This application claims the rights of US Provisional Application No. 60 / 148,848 (filed August 12, 1999). The entire disclosure of this is incorporated herein by reference.

COPYRIGHT NOTICE ACCORDING TO US PATENT ACT ENFORCEMENT RULE §1.71 (e) The portions of this disclosure include material that is subject to copyright protection. The owner of this copyright is
I have no objection to the facsimile reproduction by anyone of the patent documents or patent disclosures as expressed in the Patent and Trademark Office patent files or patent records, but otherwise reserve all copyright whatsoever.

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the field of enzymatic synthesis of combinatorial libraries of organic molecules using evolved enzymes. The present invention provides a library of enzymes capable of biocatalytically synthesizing a large number of organic molecule derivatives through directed evolution. Libraries of organic molecule derivatives can be screened to identify active compounds such as antibiotics and other therapeutic agents, herbicides and pesticides.

Background In the process of drug discovery, lead compound optimization represents one of many trials. Frequently, lead compounds lack some of the pharmacological properties required for a fully functional drug (eg, high potency, selectivity, low toxicity, bioavailability, etc.). Therefore, further modification of the lead compound is often necessary to achieve an optimized drug with the perfect combination of desired properties. Traditional approaches to derivatization rely on many empirical experiences to guide the medicinal chemist in choosing which chemical analogs to synthesize and test. Some compounds are selected for synthesis and others are not selected.
Similarly, when using combinatorial chemistry to make derivatives of lead compounds, particular building blocks are chosen for the parallel synthesis of many analogs. Other building blocks are not selected. These choices generally provide guidance on the main part of experience in medicinal chemistry, which is for modifications that would lead to improvements, and to modifications that would lead to new undesirable or exacerbated properties. Can be provided) thus made. Unfortunately, however, the bulk of this experience is unique to the individual medicinal chemist, as it is largely not fully described, except in fractional forms in numerous books.

The improvement of lead compounds with potential for pharmaceutical use is not the only situation where the derivatization of organic molecules is the goal. Organic molecules have many uses (eg, pesticides,
(Including herbicides). In order to obtain compounds that exhibit improved properties for a particular application, it is often desirable to generate a library of organic molecule derivatives that can subsequently be screened to identify derivatives that exhibit the desired properties.

Combinatorial synthetic methods have the potential to provide a method for synthesizing a wide variety of lead compound derivatives without the need for a priori assumption about which derivative is probably the most advantageous. Have. Instead of individually synthesizing the derivatives and testing the derivatives, many different derivatives can be made simultaneously. Combinatorial synthesis is not only for derivatization of lead compounds,
It is also useful in the synthesis of compounds that are screened to identify potential lead compounds that deserve further study. However, the synthesis of combinatorial libraries of organic molecule derivatives is very limited. This is because many types of organic molecule derivatives are difficult or even impossible to synthesize by pure chemical means.

Enzymes provide a potentially attractive route for the synthesis of compound libraries that can identify compounds that exhibit desired properties. Enzymes can act on a mixture of complex molecules in solution and catalyze the synthesis of derivatives of that molecule without the formation of by-products. Conventional chemical processes for lead compound derivatization are typically non-selective and require multiple protection and deprotection steps, but such steps are related to enzymatic synthesis. Not necessary. In addition, the enzyme can function under relatively mild conditions that are not destructive to the reaction products. In addition, the enzyme may make several different types of modifications to organic molecules, such as existing and potential lead compounds, and other biologically active molecules of interest. For example, the enzyme may catalyze the addition of certain moieties to the compound, such as by ester, amide, carbonate, carbamate or glycosidic linkages. The enzyme may also add new functional groups to the organic molecule or modify existing functional groups present on the compound. Enzymatic biocatalysts also have certain additional benefits (eg,
Substrate selectivity, stereoselectivity, regioselectivity).

[0008] Enzymatic combinatorial biocatalysts have great potential, but important weaknesses remain. For example, a wide variety of enzymes that can facilitate derivatization of a whole range of organic molecules are not yet available. One of ordinary skill in the art is unlikely to be able to obtain from a set of naturally occurring enzymes an enzyme that possesses the desired substrate, stereo or regiospecificity for any particular organic molecule of interest. Therefore, there is a need for derivatizing enzymes that can produce a wide variety of organic molecule derivatives. The absence of such enzymes limits the number and type of organic molecule derivatives that can be obtained by combinatorial biocatalysis. Therefore, there is a need for methods to obtain derivatizing enzymes that catalyze a wide variety of different organic molecule derivatizations, as well as libraries of such organic molecule derivatives. The present invention meets these and other needs.

SUMMARY OF THE INVENTION The present invention provides a method for obtaining a library of organic molecule derivatives. The method involves contacting an organic molecule with one or more members of a library of recombinant derivatizing enzymes and other necessary reactants to form a library of organic molecule derivatives. This derivatizing enzyme catalyzes the following reactions: a
) Modification of one or more functional groups present on the organic molecule; b) Chemical moieties on one or more functional groups present on the organic molecule.
Or c) introducing a new functional group onto the organic molecule. The method is useful for a wide variety of organic molecules, including, for example, those with pharmacological, herbicidal, insecticidal or other activities, or for industrial processes. .

[0010] In some embodiments, the method further comprises performing one or more additional reactions on the derivative obtained by contacting with the derivatizing enzyme. Therefore, the product of the first reaction acts as an intermediate for further reactions. Further reactions include, for example, adding a library of organic molecule derivatives to one or more members of the second library of recombinant derivatizing enzymes and other necessary reactants, and further libraries of organic molecule derivatives. The step of contacting to form may be included. Alternatively, this intermediate can be chemically modified or modified with other enzymes.

The library of recombinant derivatizing enzymes is, in some embodiments, obtained by: (1) recombinant at least a first form and a second form of a nucleic acid encoding a derivatizing enzyme. Recombination to produce a library of polynucleotides, wherein the first and second forms differ from each other in two or more nucleotides; and (2) live of the recombinant polynucleotides. Expressing the rally to obtain a library of recombinant derivatizing enzymes. If desired, the method can further include the following steps: (3) encoding at least one recombinant polynucleotide encoding a member of the library of recombinant derivatizing enzymes described above and derivatizing enzymes. A further form of the nucleic acid which is the same as or different from the first and second forms and is recombined to produce a further library of recombinant nucleic acids; (4) this set. Expressing an additional library of recombinant polynucleotides to obtain an additional library of recombinant derivatizing enzymes; and (5) if necessary, the additional library of recombinant derivatizing enzymes is different in the desired number of different Repeating (3) and (4) until including a recombinant derivatizing enzyme.

The present invention also provides a method for obtaining an enzyme that catalyzes the synthesis of a desired organic molecule derivative. These methods involve contacting an organic molecule with members of a library of recombinant derivatizing enzymes and other necessary reactants to form a library of organic molecule derivatives; the library of organic molecule derivatives. Identifying a desired organic molecule derivative in; and identifying members of a library of recombinant derivatizing enzymes that catalyze the synthesis of the desired organic molecule derivative.

The present invention also provides a library of recombinant derivatizing enzymes, wherein the recombinant derivatizing enzymes, when contacted with an organic molecule having one or more functional groups, are as follows: Catalyzing such reactions: a) modification of one or more of its functional groups;
b) addition of chemical moieties to one or more of its functional groups; or c) introduction of new functional groups.

In another embodiment, the invention provides a library of organic molecule derivatives. The library is biocatalytically synthesized by contacting an organic molecule having one or more functional groups with multiple members of a library of recombinant derivatizing enzymes, the recombinant derivatizing enzymes being: Catalyze a reaction such as: a) modification of one or more of its functional groups; b) addition of chemical moieties to one or more of its functional groups; or c) introduction of new functional groups. .

Detailed Description Definitions A “derivatizing enzyme” is an enzyme capable of catalyzing a reaction on an organic molecule. For example, the derivatizing enzyme may modify an existing functional group present on the molecule, add a chemical moiety on the functional group, or add a new functional group on the organic molecule. obtain. The organic molecule can include both synthetic compounds (including, for example, non-naturally occurring compounds such as halo-containing compounds) and naturally occurring compounds.

A “recombinant derivatizing enzyme” is a non-naturally occurring derivatizing enzyme that differs in sequence from at least one naturally occurring derivatizing enzyme by at least one amino acid residue. Recombinant derivatizing enzymes include derivatizing enzymes that are composed of multiple amino acid blocks, which blocks are not contiguous in naturally occurring enzymes. This block is generally of random length. The recombinant derivatizing enzyme can be chimeric,
Thus, it has a sequence portion derived from the sequences of at least two different parent enzymes. The chimeric recombinant derivatizing enzyme is encoded by a chimeric gene that includes a nucleic acid segment derived from at least two different parent genes or parent gene segments.
The parent gene may optionally encode a derivatizing enzyme.

As used herein, the term “library” refers to a collection of diverse molecules such as recombinant derivatizing enzymes and organic compound analogs. The libraries of the invention have at least two different member molecules, but can vary in size. Typically, the libraries of the invention will have at least about 5 different member, and more typically have at least about 10 different member molecules. Larger libraries of the invention typically have at least about 100 different member molecules, and sometimes more than about 10,000,
Or even have more than about 100,000. Very large libraries of the invention can have more than about 1,000,000 members.

“Functional group” refers to the atom or group of atoms that defines the structure of a particular family of organic compounds and determines their properties. For example, functional groups include alkenes, alkynes, aromatics, halogens, hydroxyls, ethers, esters, aldehydes, ketones, carboxylic acids, amides, amines and the like.

A “lead compound” is a prototype compound that has a desired biological or pharmacological activity, but may have other characteristics that are not desirable. For example, the lead compound may be toxic, insoluble, have other biological activities, or have less than optimal bioavailability (eg, absorption, distribution,
It may have metabolism and excretion (ie ADME), or it may have less than optimal biological activity, etc.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides, and polymers thereof, in either single-stranded or double-stranded form. The term refers to known nucleotide analogs or modified backbone residues or bonds (these are
Synthetic, naturally-occurring or non-naturally occurring, which have similar binding properties to the reference nucleic acid and which are metabolized in a manner similar to the reference nucleotide). Including. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNA), and the like.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as those explicitly shown. Including. The term “nucleic acid” is used herein to refer to “gene”, “cD
"NA", "mRNA", "oligonucleotide", and "polynucleotide"
Used interchangeably with.

The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residue is an analog or mimetic of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified (eg, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs are compounds (eg, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium) that have the same basic chemical structure as a naturally occurring amino acid (ie, an α-carbon that binds hydrogen, a carboxyl group, an amino group, and an R group). ). Such analogs have modified R groups (eg, norleucine) or have a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids are referred to herein by either their commonly known three letter symbols or by the IUPAC-IUB Biochemical Nomenclature.
Reference may be made by any of the one-letter symbols recommended by the Commission. Similarly, a nucleotide may be referred to by its commonly accepted one letter code.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. A conservatively modified variant, with respect to a particular nucleic acid sequence, refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or essentially if the nucleic acid does not encode an amino acid sequence. To the same sequence. In particular, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. Can be achieved by (Batzer et al., Nucleic
Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Am. B
iol. Chem. 260: 2605-2608 (1985); Rossoli.
ni et al., Mol. Cell. Probes 8: 91-98 (1994)). Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at all positions where alanine is specified by a codon, this codon can be changed to any of the described codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation of this nucleic acid. Those skilled in the art will understand that each codon (AUG (which is (
(In some organisms, along with GUG, usually the only codon for methionine), and TGG (which is usually the only codon for tryptophan) have been modified and functionally It can give rise to identical molecules. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is included in each described sequence.

With respect to amino acid sequences, individual substitutions, deletions or additions to nucleic acid sequences, peptide sequences, polypeptide sequences or protein sequences, which alter, add single amino acids or a small proportion of amino acids in the encoded sequence, Those skilled in the art will recognize that this alteration is a "conservatively modified variant" if this alteration results in the replacement of the amino acid by a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are polymorphic variants of the invention, interspecies homologues,
And alleles are added and not excluded.

The term “shuffling” is used herein to refer to recombination between non-identical sequences, and in some embodiments shuffling refers to crossover or non-homologous via homologous recombination. Crossover via recombination (eg via the cre / lox system and / or the flp / frt system) may be included. Shuffling can be performed in a variety of different formats (eg, in vitro shuffling format, and in vivo shuffling format, in silico shuffling format, shuffling format using either double-stranded or single-stranded template, primer-based shuffling format). , Nucleic acid fragmentation-based shuffling formats, oligonucleotide-mediated shuffling formats (all based on recombination events between non-identical sequences, and described in more detail below or in the references). , As well as other similar recombination-based formats).

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides libraries of recombinant derivatizing enzymes that are useful for making combinatorial libraries of compounds, especially organic molecules. A library of organic molecule derivatives obtained using a recombinant derivatizing enzyme library is also provided. Libraries of organic molecule derivatives are useful, for example, for identifying derivatives having a desired biological activity, and thus are tested as lead compounds, for example, for testing pharmaceutical or other uses, and for improvement. Suitable for making combinatorial libraries of previously identified derivatives of lead compounds for testing for pharmacological or other parameters. The compound is often an organic molecule, including synthetic molecules (including, for example, non-naturally occurring compounds) and natural products (such as antibiotics).

The library of recombinant derivatizing enzymes provided by the present invention offers several advantages over previously available methods for obtaining libraries of organic molecule derivatives. For example, this recombinant library is characterized by features (catalyst rate and catalytic constant, stereospecificity, regiospecificity, enantiospecificity, multiplicity of substrate selectivity, product inhibition, in the solvent used for biocatalytic synthesis). Enzymes, which generally have catalytic properties that differ from each other, such as stability in chemical processes). Thus, the large number of different enzymes that occur increases the number of different compounds that can be produced by biocatalytic reactions. When one enzyme is used in a biocatalyst with a single organic molecule and a single chemical moiety donor, generally only one derivative is produced. In contrast, multiple recombinant enzymes appear to contain enzymes capable of catalyzing different reactions associated with the original enzyme, and thus can be initiated with the same substrates used with the original enzyme. , Can produce different products. Furthermore, the use of enzymes for the synthesis of organic compounds of interest greatly facilitates the scale-up of synthetic reactions.

In a presently preferred embodiment, DNA shuffling or other methods of recursive recombination are used to generate a library of recombinant enzymes. DNA shuffling has proven very effective in improving the level of known activity of biocatalysts. A further value of this technique lies in the ability to generate catalytic activity previously unknown among wild-type enzymes. Thus, this technique provides a reliable means of biocatalyst production that reduces or even eliminates the need to obtain a naturally occurring biocatalyst for the targeted reaction. DNA shuffling of families of related genes, for example, produces functionally diverse gene libraries with different physical properties over more complex sequence spaces than can be found naturally for a particular protein. Since new members of these enzyme libraries have never been placed under selective pressure in organisms, these members are unbiased and rare or absent in natural samples. , Can be screened for new activity. Thus, diverse and complex enzyme libraries can be created that catalyze a fixed spectrum of important chemistries. For example, enzymes modify functional groups present on organic molecules, add chemical moieties onto functional groups (eg, acylation, glycosylation, and methylation), and introduce new functional groups into organic molecules. (For example, introduction of hydroxyl group by oxidation, introduction of double bond by reduction, etc.) can be catalyzed. Enzyme libraries can be used to directly synthesize multiple products starting from a substrate mixture or to synthesize specific compounds starting from a defined set of substrates. Alternatively, a single member of a library of recombinant enzymes can be used to synthesize a mixture of compounds by contacting that member with a substrate mixture. In a further alternative embodiment of the invention, each individual member of the library of recombinant derivatizing enzymes is tested on a defined substrate set,
Enzymes with novel and useful substrate selectivity, or other useful characteristics can be identified.

The organic molecule derivatives thus synthesized can then be screened to identify those with the desired properties, or further modified by one or more additional chemical or enzymatic reactions. obtain. Also, enzyme libraries are screened to identify those enzymes with novel and useful substrate selectivity, or other desired characteristics, and to use these enzymes to produce the desired compounds. Can be done.

The recombinant enzyme obtained using the method of the present invention can be used in vitro or expressed by microbial cells which carry out the biocatalysis. In some embodiments, the microorganism is modified to express one or more derivatizing enzymes for efficient biocatalytic production of derivatized products. For example, the microorganism can include one or more recombinant polynucleotides that encode an improved acyl transferase, glycosyl transferase, oxidase, methyl transferase, or other biocatalytic enzyme, which are then expressed by the microbial cell. These polynucleotides can be introduced into organisms that naturally produce the desired starting substrate.
For example, a polynucleotide encoding a recombinant derivatizing enzyme is a polyketide (p
can be introduced into an organism that naturally produces or has been engineered to produce. Thus, a recombinant polynucleotide encoding a recombinant derivatizing enzyme of the present invention can be used in organisms that biosynthesize the backbone of organic molecules for in vivo derivatization of organic compounds whose backbone was previously prepared. Useful for in vivo derivatization of organic molecules and for in vitro use to derivatize previously prepared organic molecules.

A. Generation of Recombinant Libraries The present invention, in some embodiments, involves the production of recombinant libraries of polynucleotides. This library is then screened for desired properties (eg, enhanced enzymatic activity, enhanced stereospecificity, enhanced regiospecificity,
And those libraries encoding enzymes or other polypeptides that exhibit enhanced enantiospecificity, reduced sensitivity to inhibitors, processing stability (eg, solvent stability, pH stability, thermostability, etc.). Identify members. Recombinant libraries can be made using any of a variety of methods, including those described herein. For example, various nucleic acid shuffling protocols are available and are well described in the art. The following publications disclose various such procedures and / or methods that may be incorporated into such procedures,
As well as a variety of other production protocols.

[0034]

[Table 1] Further details regarding the DNA shuffling method are found by the inventors and their co-workers in US patents including:

[0035]

[Table 2] Further details and formats for DNA shuffling protocols are found in various PCT publications and foreign patent application publications, including:

[0036]

[Table 3] Certain US applications, including the following, provide further details regarding DNA shuffling and related techniques, as well as other diversity generation methods.

[0037]

[Table 4] The shuffling format using a single-stranded template is described below.

[0038]

[Table 5] As the reviews of the aforementioned publications, patents, published applications, and U.S. patent applications reveal, shuffling of nucleic acids to provide new nucleic acids with the desired properties has led to many well-established recombinants. Methods, and these procedures may be combined with any of a variety of other diversity generation methods.

Briefly, several different general classes of recombination methods are applicable to the present invention and are set forth in the references above. First, the nucleic acid is in vitro by any of the various techniques discussed in the above references, including, for example, DNAse digestion of the nucleic acid to be recombined, followed by ligation and / or PCR assembly of the nucleic acid molecule. Can be recombined with. Second, nucleic acids can be recursively recombined in vivo, for example by causing recombination between nucleic acids in cells. Third, whole genome recombination methods can be used. Here, the entire genome of the cell or other organism is recombined, optionally including spiking of the genomic recombination mixture with the desired library components. Fourth, synthetic recombination methods can be used. In this method, oligonucleotides corresponding to a target of interest are synthesized and reassembled in a PCR or ligation reaction containing oligonucleotides corresponding to more than one parent nucleic acid, thereby producing new recombinant nucleic acids. Oligonucleotides can be made by standard nucleotide addition methods or can be made by trinucleotide synthetic approaches. Fifth,
In silico recombination methods can be accomplished. In this method, a genetic algorithm is used in a computer to analyze nucleic acid homologs (
Alternatively, a sequence string (sequence st) corresponding to a heterologous sequence)
ring). The resulting recombined sequence string is optionally converted into a nucleic acid by, for example, partnering with an oligonucleotide synthesis / gene reassembly technique to synthesize the nucleic acid corresponding to the recombined sequence. To do. Any of the common recombination formats described above can be performed in a recursive manner to produce a more diverse set of recombinant nucleic acids. Sixth, methods of approaching natural diversity can be used, for example, hybridization of a variety of nucleic acids or nucleic acid fragments to a single-stranded template, followed by polymerization and / or ligation to regenerate the full length sequence. , Optionally, then by degradation of the template and recovery of the resulting modified nucleic acid.

To illustrate, in one embodiment of the invention, the shuffling method is used to prepare a polynucleotide encoding a recombinant derivatizing enzyme. This method includes: Overlapping a population of variant polynucleotides under conditions in which one segment serves as a template for the extension of another segment to produce a population of recombinant polynucleotides. Initiating the polynucleotide amplification process on the desired segment; and selecting or screening the recombinant polynucleotide for the desired property.

Overlapping segments can be generated by various methods as described or referred to herein (eg, chemical synthesis, cleavage or fragmentation of a population of polynucleotides,
Amplification, and other methods well known in the art).

In another embodiment, the shuffling method used to produce the recombinant derivatizing enzyme comprises: a step of hybridizing at least two nucleic acid sets, wherein the first A nucleic acid set comprising a single stranded nucleic acid template and a second nucleic acid set comprising at least one nucleic acid fragment set; and extending or linking a sequence gap between the hybridized nucleic acid fragments. , Or both to produce at least substantially full-length chimeric nucleic acid sequence corresponding to the single-stranded nucleic acid template, thereby recombination of the set of nucleic acid fragments, and, optionally, at least Substantially full-length chimeric nucleic acid sequence and denaturing this single-stranded nucleic acid template Separating the at least substantially full length chimeric nucleic acid sequence from the single stranded nucleic acid template by at least one separation technique; and separating at least substantially by nuclease digestion or physical fragmentation; Fragmenting the full-length chimeric nucleic acid sequence to provide a chimeric nucleic acid fragment.

The above references provide these and other basic recombinant formats, as well as many variations of these formats. Regardless of the shuffling format used, the nucleic acids of the invention can be recombined (with each other, or with related or even unrelated) to produce a diverse set of recombinant nucleic acids (eg, homologous nucleic acid sets). Can be included).

After recombination, any nucleic acid produced may be selected for the desired activity. In the context of the present invention, this may involve testing for and identifying any activity that can be detected in an automated format by any assay in the art. A variety of related (or even unrelated) properties can be assayed using any available assay. These methods are automated in accordance with the present invention as described herein.

Mutagenesis and shuffling of DNA provides a robust and universally available means of producing useful diversity in manipulating proteins, pathways, cells and organisms with improved characteristics. In addition to the basic form described above, it is sometimes desirable to combine shuffling methods with other techniques for generating diversity. In combination (or separately) with the shuffling method, various diversity generation methods can be performed and the results (ie, the diversity population of nucleic acids) screened in the system of the invention. Further diversity can be introduced by mutagenesis methods known in the art.

[0046]   Examples of the mutagenesis method include the methods described below.

[0047]

[Table 6] Further suitable methods include:

[0048]

[Table 7] More details on many of the above methods can be found in Methods in Enzym.
ology, vol. 154. It also describes controls useful for troubleshooting problems associated with various mutagenesis methods.

Kits for mutagenesis are commercially available. For example, the kit is available from

[0050]

[Table 8] Moreover, any of the shuffling techniques described can be used in combination with procedures that introduce additional diversity into the genome (eg, bacterial genome). For example,
Techniques have been proposed for producing nucleic acid multimers suitable for transformation into various species, including E. coli and B. subtilis (eg Schelle).
nberger, US Pat. No. 5,756,316). Consist of genes that differ with respect to each other (eg, derived from natural diversity or via site-directed mutagenesis, error-prone PCR, application of passage through mutagenic bacterial strains, etc.) Transforming such a multimer into a suitable host,
Introduce an additional source of nucleic acid diversity for DNA shuffling. Multimers transformed into host species are particularly suitable as substrates for in vivo shuffling protocols. Alternatively, various 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 a library, each of which is a member of the library containing a single homologous population of monomeric or pooled nucleic acids. Alternatively, the monomeric nucleic acid can be recovered by standard techniques and recombined in any of the shuffling formats described.

A shuffling format using the chain termination method has also been proposed (see, eg, US Pat. No. 5,965,408). In this approach, double-stranded DNA corresponding to one or more genes sharing regions of sequence similarity
In the presence or absence of primers specific for this gene,
Then degenerate. The single-stranded polynucleotide is then combined with a polymerase and chain terminating reagents (eg, UV, gamma, or X-ray irradiation; ethidium bromide or other intercalating agent; DNA binding protein (eg, single-stranded binding protein , Transcriptional activators, or histones; polycyclic aromatic hydrocarbons;
Annealing and incubating in the presence of trivalent chromium or trivalent chromium salts; or abreviated polymerization, such as mediated by rapid thermal cycling, resulting in formation of partial double-stranded molecules . The partially double-stranded molecule (eg, including the partially extended chain) is then denatured and reannealed in subsequent rounds or partial replications, sharing varying degrees of sequence similarity, And yields a polynucleotide that is chimeric with respect to the starting population of DNA molecules. If desired, the product or partial pool of products can be amplified at one or more stages of the process. Polynucleotide products produced by the chain termination method, as described above, are suitable substrates for further DNA shuffling according to any of the described formats.

Diversity uses a non-homologous-based shuffling method, which is described in the publications above, and the application may be homologous or non-homologous based, depending on the exact format. Can be further increased by For example, Oster
meier et al. (1999) “A combinatorial approac
h to hybrid enzymes independent of D
NA homology "Nature Biotechnology. 17: 120
Growth cleavage (i) for the production of the hybrid enzyme (ITCHY) described in 5.
ncremental truncation can be used to generate shuffled libraries, which can optionally serve as substrates for one or more in vitro or in vivo shuffling methods. Ostermeier et al. (1999), “Combinatorial pr
otain engineering by incremental true
", Proc. Nat'l. Acad. Sci. USA 96:
3562-3567; Ostermeier et al. (1999), "Increme.
ntal truncation as a strategy in the
engineering of novel biocatalysts ",
Biological and Medicinal Chemistry, 7
See also: 2139-2144.

Methods for producing multiple expression libraries have been described (eg, US Pat. Nos. 5,783,431; 5,824,485) and for identifying protein activities of interest. Its use has been proposed (US Pat. No. 5,958).
, 672). A polymorphic expression library is generally a library containing cDNA or genomic sequences from multiple species or strains operably linked to appropriate regulatory sequences in an expression cassette. The cDNA and / or genomic sequences are optionally randomly linked to further increase diversity. The vector may be a shuttle vector suitable for transformation and expression in more than one species of host organism (eg bacterial species, eukaryotic cells). In some cases, the library is biased by preselecting sequences that encode the protein of interest or hybridize to the nucleic acid of interest. Any such library can serve as a substrate for any of the shuffling methods described herein.

In some applications, the library (eg, amplified library,
Preselection or prescreening of a genomic library, a cDNA library, a normalization library, etc.) or other substrate nucleic acid prior to shuffling, or biasing the substrate to a nucleic acid encoding a functional product. (The shuffling procedure may also have these effects independently).
For example, in the case of antibody engineering, any of the described methods can bias the shuffling process against antibodies having a functional antigen binding site by utilizing an in vivo recombination event prior to DNA shuffling. is there. For example, recombinant CDRs from a B cell cDNA library can be amplified and assembled into framework regions prior to DNA shuffling according to any of the methods described herein (eg, Jirholt et al. (1998)
"Exploiting sequence space: shuffling
in vivo formed complementarity dete
mining regions into a master frame
ork "Gene 215: 471).

The library can be biased for nucleic acids that encode proteins that have the desired enzymatic activity. For example, after identifying a clone from a library that exhibits a specified activity, the clone may be cloned using any known method for introducing DNA changes, including but not limited to DNA shuffling. It can be mutagenized. The library containing the mutagenized homologs is then subjected to the desired activity, which can be the same as the initial activity specified, or different.
To be screened for. An example of such a procedure is described in US Pat. No. 5,939.
, 250. The desired activity can be identified by any method known in the art. For example, WO 99/10539 screens a gene library by combining extracts from the gene library with components obtained from metabolically enriched cells and identifying combinations that exhibit the desired activity. Suggest to get. A clone having the desired activity introduces a bioactive substrate into a sample of the library, and a bioactive fluorescence corresponding to the product of the desired activity is measured by a fluorometer (eg, flow cytometry device, CCD).
It has also been proposed (eg WO98 / 58085) that it can be identified by detection using a fluorimeter, a fluorometer, or a spectrophotometer).

The library can also be biased against nucleic acids having specific characteristics, such as hybridization to selected nucleic acid probes. For example, application WO 99/10539 describes a desired activity (eg enzymatic activity, eg: lipase,
A polynucleotide encoding an esterase, protease, glycosidase, glycosyltransferase, phosphatase, kinase, oxygenase, peroxidase, hydrolase, hydratase, nitrilase, transaminase, amidase or acylase) can be identified from the genomic DNA sequence in the following manner: To propose. Single-stranded DNA molecules from a population of genomic DNA are hybridized to the ligand-conjugated probe. This genomic DNA is
It can be either from cultured or uncultured microorganisms or from environmental samples. Alternatively, the genomic DNA can be from a multicellular organism, or tissue derived from the multicellular organism.

The synthesis of the second strand can be carried out directly from the hybridization probe used in the capture, with or without pre-release from the capture medium, or in the art. It can be performed by a wide variety of other strategies known in the art. Alternatively, the isolated single-stranded genomic DNA population can be fragmented without further cloning and used directly in shuffling format utilizing a single-stranded template. Several single-stranded template shuffling formats have been described, for example, in WO98 27239, "ME.
THEDS AND COMPOSITIONS FOR POLYPEPTI
DE ENGINEERING ", Patten et al .;" SINGLE STRA "
NDED NUCLEIC ACID TEMPLATE-MEDIATED
RECOMBINATION AND NUCLEIC ACID FRAGM
ENT ISOLATION ", Affholter, USSN 60/186
, 482 (filed on March 2, 2000); "METHODS FOR GENERATOR
ATING HIGHLY DIVERSE LIBRARIES ", WO 0
000632; and "METHOD FOR OBTAINING IN V
ITRO RECOMBINED POLYNUCLEOTIDE SEQUE
NCE BANKS AND RESULTING SEQUENCES ", W
O 0009679. In one such method, the genomic library-derived fragment population is annealed with partial or often near full length ssDNA or RNA, which correspond to the opposite strand. The assembly of complex chimeric genes from this population is mediated by nuclease-based removal of non-hybridizing fragment ends, polymerization to fill the gap between such fragments, and subsequent single-stranded ligation. The parental strands are removed by digestion (if containing RNA or uracil), magnetic separation under denaturing conditions (if labeled in a manner that leads to such separation), and other available separation / purification methods. obtain. Alternatively, the parental strand is optionally co-purified with the chimeric strand and removed during subsequent screening and processing steps.

In the conventional approach, single-stranded molecules are converted to double-stranded DNA (dsDNA) and dsDNA molecules are attached to a solid support by ligand-mediated conjugation. After separation of unbound DNA, the selected DNA molecules are released from the support and introduced into a suitable host cell to create a library-enriched sequence that hybridizes to this probe. The library produced in this manner provides the desired substrate for further shuffling using any of the shuffling reactions described herein.

It is also possible that any of the techniques described above suitable for enriching the library prior to shuffling can be used to screen the products generated by the method of DNA shuffling. To be understood.

In a presently preferred embodiment, the recombinant library is prepared using DNA shuffling. Shuffling and screening or selection can be used to "evolve" individual genes, entire plasmids or viruses, multigene clusters, or even entire genomes (Stemmer (1
995) Bio / Technology 13: 549-553). Iterative recombination and screening / selection cycles can be performed to further evolve the nucleic acid of interest, if desired. Such techniques do not require the extensive analysis and calculations required by conventional methods for polypeptide engineering. Shuffling allows the recombination of a large number of mutations in a minimal number of screening / selection cycles, as opposed to traditional pairing recombination events. Thus, the sequence recombination techniques described herein provide that these techniques provide for recombination between mutations in any or all of these, whereby different combinations of mutations are desired. It offers particular advantages in that it provides a very fast way of exploring modalities that can influence the results. However, in some cases structural and / or functional information is available, although not required for sequence recombination, which provides an opportunity for modification of the technology.

These shuffling methods typically utilize modified forms of at least two starting nucleic acid substrates. Variant forms of candidate substrates may exhibit substantial sequence or secondary structural similarity to each other, but they should also differ in at least two positions. The initial diversity between forms may be the result of natural variation, for example different variant forms (homologues) may be obtained from different individuals or strains of organisms (including geographical variants), or It constitutes related sequences from the same organism (eg, allelic variants). Alternatively, the first diversity may be induced, eg, the second variant form may be error prone to the first variant form.
anscription) (eg, error prone (error prone
) PCR), or the use of a first variant form of a polymerase that lacks proofreading activity (see, eg, Liao (1990) Gene 88: 107-111), or a first form in a mutagenized gene line. Or by mutagenesis process of DNase fragmentation and reassembly by an error prone polymerase. The initial diversity between substrates is greatly increased in the process of subsequent recursive sequence recombination.

In a presently preferred embodiment, the shuffling of a “family” of nucleic acids comprises
Used to generate a library of recombinant polynucleotides. When a family of nucleic acids is shuffled, nucleic acids encoding homologous polypeptides from different strains, species or gene families, or portions thereof, are used as the different forms of the nucleic acids. Direct amplification of homologues with designed primers becomes more and more feasible, as genomics provide ever increasing amounts of sequence information. For example, the sequences of lipase or protease genes from several species can be considered to design primers for amplification of homologs. The resulting nucleic acid segment can then be subjected to shuffling.

All of the shuffling methods described herein can be readily utilized in the practice of the present invention. For example, in codon modification shuffling (Patter
n et al. "SHUFFRING OF CODON ALTERNED GENES"
Filed September 29, 1998 (USSN 60 / 102,362), filed January 29, 1999 (USSN 60 / 117,729), and September 2, 1999.
8 days application (detailed in USSN 09 / 102,362), the nucleic acid is
The codons that encode the polypeptide are altered and synthesized, thus allowing access to completely different mutagenic clouds upon subsequent mutation of the nucleic acid. This increases the sequence diversity of the starting nucleic acids for shuffling protocols, which modifies the speed and outcome of forced evolutionary procedures. The codon modification procedure can be used, for example, to modify any derivatizing enzyme that encodes a nucleic acid herein before performing DNA shuffling, or the codon modification approach is as described below. It can be used in combination with the oligonucleotide shuffling procedure.

Codon modified shuffling involves selecting a first nucleic acid sequence encoding a first polypeptide sequence or a portion thereof. A plurality of codon-altered nucleic acid sequences, each of which encodes a portion or all of the first polypeptide, or a modified or related polypeptide is then selected (eg, a library of codon-altered nucleic acids is Can be selected in a biological assay that recognizes a component or activity of the library), and a plurality of codon-altered nucleic acid sequences to produce a target codon-altered nucleic acid encoding part or all of a second protein. Can be replaced. The target codon-altered nucleic acid is then screened for detectable functional or structural properties, optionally including comparison with properties of the first and / or related polypeptides. The purpose of such a screen is to identify polypeptides that have structural or functional properties that are equivalent to or superior to the first polypeptide or related polypeptides. Nucleic acids encoding such polypeptides can be altered by essentially any desired procedure (cell, vector, virus (eg, as a component of a vaccine or immunogenic composition), transgenic organism, etc.). (Including introducing a nucleic acid).

“In silico” shuffling (Selifonov)
And Stemmer, “METHODS FOR MAKING CHARA
CTER STRINGS, POLYNUCLEO TIDE & POLYPEPT
IDES HAVING DESIRED CHARACTERISTICS "
Filed February 5, 1999 (USSN 60,118,854) and 1999
Filed on October 12, 2010 (described in detail in USSN 09 / 416,375)
Utilizes a genetic algorithm in a computer to utilize a computer algorithm to perform "virtual" shuffling. As applied to the present invention, the derivatizing enzyme gene sequence string is recombined in a computer system and the desired product is made (eg, by reassembly PCR of synthetic oligonucleotides). Briefly, genetic manipulations (algorithms that represent a given genetic event (eg, point mutation, recombination of two strands of homologous nucleic acids, etc.)), for example, align nucleic acid sequence strings (standard alignment). Software or by manual inspection and alignment), and by predicting recombination results, to model possible recombination or mutation events in one or more nucleic acids. The expected recombination result is
Used to produce corresponding results. This expected recombination result produces the corresponding molecule (eg, oligonucleotide synthesis and reassembly PC
R)).

“Oligonucleotide-mediated shuffling” (Crameri et al., “OLIGO
NUCLEOTIDE MEDIATED NUCLEIC ACID REC
OMBINATION ”, filed February 5, 1999 (USSN 60/118,
813) and filed June 24, 1999 (USSN 60 / 141,049)
, And in the application filed Sep. 28, 1999 (USSN 09 / 408,392), corresponding oligonucleotides to a family of related homologous nucleic acids (eg, encoding a nucleic acid when applied to the present invention). Interspecific or allelic variants of derivatizing enzymes) are recombined to produce selectable nucleic acids.

One advantage of oligonucleotide-mediated recombination is the ability to recombine homologous nucleic acids with low sequence similarity, or even heterologous nucleic acids. In these low homology oligonucleotide shuffling methods, one or more sets of nucleic acid segments are recombined using, for example, a set of cross family diversity oligonucleotides. Each of these crossover oligonucleotides has multiple sequence diversity domains, corresponding to multiple sequence diversity domains from homologous or heterologous nucleic acids with low sequence similarity. Crossover oligonucleotides derived by comparison with one or more homologous or heterologous nucleic acids can hybridize to one or more regions of a nucleic acid segment, which facilitates recombination.

When recombining homologous nucleic acids, a set of overlapping families of oligonucleotides, which are derived by comparison of homologous nucleic acids and synthesis of oligonucleotide segments, are hybridized and extended (eg, reassembly PCR). ), Thereby providing a population of recombinant nucleic acids, which population can be selected for a desired trait or characteristic. Typically, sets of overlapping oligonucleotides are subsequences of consensus regions from multiple homologous target nucleic acids.
ce)). In general, sets of overlapping oligonucleotides are provided by aligning homologous nucleic acid sequences to select conserved regions of sequence identity and regions of sequence diversity. Multiple oligonucleotides have been synthesized (sequentially or in parallel), which correspond to at least one region of sequence diversity.

A set of segments, or a subset of segments, used in the oligonucleotide shuffling approach is by cleavage of one or more homologous nucleic acids (eg, with Dnase), or, more commonly, of at least one nucleic acid. It can be provided by synthesizing a set of oligonucleotides corresponding to multiple regions, typically the oligonucleotide corresponding to a full length nucleic acid is provided as a member of a set of nucleic acid fragments. In the shuffling procedure described herein, these segments (eg, segments of a derivatizing enzyme that encodes a nucleic acid) are linked to produce a shuffling family of oligonucleotides (eg, a recombinant derivatizing enzyme that encodes a nucleic acid). In one or more recombination reactions).

Often, improvements are achieved after one round of recombination and screening / selection. However, recursive sequence recombination can be used to achieve even further improvements in the desired properties. Sequence recombination can be accomplished in many different forms and permutations, which share some common principles. Recursive sequence recombination involves successive cycles of recombination to produce molecular diversity. That is,
It produces a family of nucleic acid molecules that show some degree of sequence identity with each other but differ in the presence of a mutation. In any given cycle, recombination can occur in vivo or in vitro, intracellularly or extracellularly. Moreover, the diversity resulting from recombination can be cycled by applying prior art methods of mutagenesis (eg, error-prone PCR or cassette mutagenesis) to either substrates or products for recombination. Can be increased in. In some cases, a new or improved property or characteristic is a variant form of a sequence that is a homologue from a strain of a different individual or organism, or a related sequence from the same organism, such as an allelic variant. If used, it can be achieved after only one cycle of in vivo or in vitro recombination.

Expression of the recombinant polynucleotide to obtain the recombinant derivatizing enzyme is generally accomplished in cells. A library of recombinant polynucleotides can be produced either in vitro or in vivo, as described in US Pat. No. 5,837,458. Thus, for the production of in vitro libraries, recombinant polynucleotides are introduced into cells for expression.

B. Types of Derivatizing Enzymes Useful for Biocatalytic Synthesis of Combinatorial Libraries The method of the present invention is applicable to a wide range of derivatizing enzymes that can catalyze the modification of organic molecules of interest. Such an enzyme may modify the substrate by, for example, adding a functional group to the molecule or modifying a functional group present on the molecule. Modification of interest also includes the addition of chemical moieties to functional groups. In a presently preferred embodiment, the derivatizing enzyme does not add to the backbone length of the organic molecule. The type of reaction of interest is, for example,
Khmelnitsky et al. (1996) Molecular Diversit
y and Combinatorial Chemistry, Chapter
r 14, pp. 144-157 (American Chemical So
City), and Michels et al. (1998) Tibtech 16:
210-215. Different types of derivatizing enzymes and examples of application of the method of the invention to these enzymes are described below.

In addition to the increased diversity of enzymatic activity found in libraries of recombinant enzymes, organic compounds (eg, natural compounds, non-natural compounds (eg, 5-fluorouracil, azidothymidine, etc.), small molecules, And polymers (eg peptides and peptide variants, oligonucleotides / polynucleotides and variants thereof, polyhydroxyalkanoates, polysaccharides, polylactic acid, polylactic acid-
Enzymes with increased specific properties are available which increase the utility of the enzyme in modifying co-glycolic acid, polyethylene glycol), etc.). Small molecules used in the practice of the present invention typically have a molecular weight of less than about 2500 daltons, usually less than about 2000 daltons, and sometimes less than about 1500 daltons.

These libraries were identified in order to identify members of these libraries which encode enzymes which show an improvement in the desired properties for use in the reaction of interest as compared to the wild type enzyme. Can be screened. For example, screening to identify members of these libraries that encode enzymes with improved substrate specificity for a particular compound, or improved regioselectivity at desired functional groups on the compound. obtain.

In some embodiments, the library of recombinant derivatizing enzymes is a variant of a given wild-type gene, into which a method of generating diversity (eg, described herein). Mutations are introduced by methods such as the shuffling and gene reassembly shuffling process. Thus, a limited but complete diversity can be provided around a given array using dense sampling. In other embodiments, recombinant libraries are produced by applying the diversity generation method to several different wild-type genes. Limited and incomplete diversity is achieved, which scatters throughout the functional array space, as in sparse sampling. This latter technique is preferred when generating new enzyme specificity.

1. Modification of Existing Functional Groups and Introduction of New Functional Groups into Organic Molecules In some embodiments, the recombinant derivatizing enzyme, and libraries thereof, may contain a target organic compound such as a lead compound. It can catalyze the modification of existing functional groups present on the molecule. For example, the derivatizing agent of interest may oxidize or reduce functional groups, hydrolyze groups, or replace one functional group with another. Other reactions of interest include lactonization, isomerization, and epimerization.

A. Hydroxylation In some embodiments, hydrogens in organic molecules are replaced with hydroxyl groups. This can often result in marked changes in biological activity. Hydroxylation is often associated with increased metabolism due to first passage through the liver. The introduction of hydroxyl groups in drug candidates may also confer a more rapid metabolism by the action of subsequent group transfer enzymes, such as enzymes that catalyze methylation, sulfation, phosphorylation and glycosylation.

Among the derivatizing enzymes, the enzymes useful for introducing the hydroxyl group are monooxygenases and dioxygenases. A range of monooxygenases known in the art provide suitable starting points for making libraries of recombinant monooxygenases useful in the methods of the invention. One useful class of monooxygenases is the heme-dependent eukaryotic and bacterial cytochrome P-45.
It is exemplified by 0. In the presence of oxygen and an intact redox recycling system, P450 exhibits monooxygenase activity. However, the addition of hydrogen peroxide or other peroxides can be used to circumvent the NAD (P) H requirement (ie, allowing peroxidase activity) for many of the same substrates. P4
The ability of enzymes such as 50 to carry out chemical reactions at chemically distinct sites is well known.
Steroid modification by naturally occurring P450s is pervasive in biosynthesis and drug metabolism. Thus, for example, a shuffled library of P450s will generate many new attachment points for further chemistry (or enzymatic derivatization) or screening. The other enzyme classes mentioned herein also have utility in generating new structural diversity in clinically important families of compounds.

The P450 monooxygenase gene family is particularly well adapted for the use of family shuffling to obtain recombinant derivatizing enzymes. About 70-80 families of P450 monooxygenases from many different species are known. For the identification of homologous genes that can be shuffled together as a family, a representative alignment of P450 enzymes is the CYTOCHROME P450
: STRUCTURE, MECHANISM, AND BIOCHEMISTR
^{Y, 2 nd Addition (Paul R.Ortiz} de Montel
lano) Plenum Press, New York, 1995) ("O
rtiz de Montellano ").
The latest list of P450s can be found on the World Wide Web (http: // drnel
son. utmem. edu / homepage. html) can be found electronically. To illustrate the application of shuffling to improve the family of P450 enzymes, one or more of more than 1000 members of this superfamily were selected, aligned with similar homologous sequences, and their homologues. Shuffled to a different array. For example, the gene for the bovine P450 _scc enzyme (CYP11A1) belongs to a family closely related to the P450 gene. DNA shuffling (Crameri et al., Nature)
391: 288) can be used to generate hybrid variants from this family of genes, and this library can be used to make combinatorial libraries of organic molecule derivatives. In particular, Streptomyces produces P450 monooxygenase which is used in the production of natural products such as antibiotics. Examples of P450 monooxygenase genes suitable for shuffling include the following, each of which is at least 45% identical at the amino acid level: cytochrome p450 monooxygenase (S. venezuelae) AF0.
87022 Cytochrome p450 monooxygenase (Sac. Erythraea) M8
3110 cytochrome p450 monooxygenase (Sac. Erythraea) M5
4983 Cytochrome p450 monooxygenase (S. hygroscopicus)
X86780 Cytochrome p450 monooxygenase (S. antibioticus) L
47200 Construction of a library of recombinant p450 monooxygenase genes was performed in co-pending, commonly assigned US patent application Ser. No. 60 / 148,850.
Filed on August 12, 1999).

It is noted that the basic chemistry described below in relation to monooxygenase is known. In addition to Ortiz de Montellano (supra),
For general guidance on various related chemistries, see Stryer (1988) BI.
OCEMISTRY, 3rd edition (or later editions) Freeman and
Co. New York, NY; Pine et al. ORGANICK C
HEMISTRY FOURTH EDITION (1980) McGraw-
Hill, Inc. (USA) (or later edition); March, ADVAN.
CED ORGANIC CHEMISTRY REACTIONS, MECH
ANISMS and Structure 4th Edition J. Wiley and
Sons (New York, NY, 1992) (or later version); Gr
eene et al., PROTECTIVE GROUPS IN ORGANIC C
Hemistry, 2nd Edition, John Wiley & Sons, New Y
ork, NY, 1991 (or later edition); Lide (ed.) (1995) T
HE CRC HANDBOOK OF CHEMISTRY AND PHY
SICS 75TH EDITION (or later editions); and in the references listed above. Further, extensive guidance on many chemical and industrial processes applicable to the present invention can be found in KIRK-OTHME.
R ENCYCLOPEDIA OF CHEMICAL TECHNOLOG
Y (3rd and 4th edition, throughout 1998), Martin Grayso
n, Executive Editor, Wiley-Interscience
e, John Wiley and Sons, NY, and among them ("Kir
k-Othmer ").

Other monooxygenase enzymes suitable for introducing hydroxyl groups and other modifications of organic molecules include monooxygenase enzymes having activities such as: alkane oxidation (eg, hydroxylation, ketones, aldehydes). Etc.), alkene epoxidation, aromatic hydroxylation, N-dealkylation (eg,
Alkylamine), S-dealkylation (eg reduced thioorganic material), O-
Dealkylation (eg of alkyl esters), oxidation of aryloxyphenols, conversion of aldehydes to acids, conversion of alcohols to aldehydes or ketones, dehydrogenation, decarbonylation, oxidative haloaromatics and halohydrocarbons. Dehalogenation, Baeyer-Villiger monoatomic oxygenation (monooxygen
cation), modification of cyclosporine, hydroxylation of mevastatin, hydroxylation of erythromycin, N-hydroxylation of sulfonylureas, sulfoxide formation or oxygenation. Other oxidative transformations will be apparent to those skilled in the art. Examples of monooxygenases suitable for use in the present invention were filed on Aug. 12, 1999, "
DNA SHUFFRING OF MONOOXYGENASE GENES
FOR PRODUCTION OF INDUSTRIAL CHEMIC
Co-pending, commonly assigned US Patent Application No. 09 entitled "ALS"
/ 373,928.

Dioxygenases are another class of derivatizing enzymes useful in the biocatalytic synthesis of organic molecule derivatives. For example, bacterial arene dioxygenase (ADO) is
The π bond can be oxidized to its corresponding vicinal diol. In the presence of oxygen and reducing compounds (eg NAD (P) H), these enzymes catalyze the reductive diatomic oxygenation of diverse compounds such as aromatic rings and non-aromatic multiple bonds. The non-phenolic nature of the ring cis dihydroxylation products resulting from the action of arene dioxygenases allows the organic by avoiding the accumulation of toxic and reactive epoxide intermediates that can significantly impair the performance of this biocatalyst. It offers significant advantages in the production of molecular derivatives.

As arene dioxygenase, for example, toluene 2,3-dioxygenase, isopropylbenzene 2,3-dioxygenase, benzene-1,2-dioxygenase, biphenyl-2,3-dioxygenase, naphthalene-1, Two
-Dioxygenases, as well as many homologous and / or functionally similar enzymes. Polynucleotides encoding suitable arene dioxygenases can be obtained from many organisms using cloning methods known to those of skill in the art.
The following list provides examples of polynucleotides that encode arene dioxygenases and that are suitable for use in the methods of the invention. The locus is Gen
Identified by Bank ID and encodes the complete or partial protein component of arene dioxygenase. Suitable loci include, for example: [PSETODC1C] toluene-1,2-dioxygenase; [
AF006691], [PJU53507], [PSECUMA], [REU2
4277], isopropylbenzene-2,3- [E04215], [PSEBD
O] dioxygenase; benzene-1,2-dioxygenase; [AEBPHA
1F], [CTU47637], [D78322], [D88020], [D8
8021], [PSEBPHA], [PSEBPHABC], [PSEBPHA]
BCC], [PSU95054], [RRBPHA1], [RGBPHA],
[RSU27591] Biphenyl-2,3-dioxygenase; [PSU152
98] Chlorobenzene dioxygenase; [AB004059], [AF010
471], [AF036940], [AF053735], and [AF053736].
], PAF079317], AF004283], [AF004284], [P
SENAPDOXA], [PSENDPOXB], [PSENDOBC],
[PSEORF1], [PSU49496] naphthalene-1,2-dioxygenase; [AF009224], [PSEBEDC12A] benzoic acid-1,2-dioxygenase; [PWWXYL] toluene dioxygenase; [ASCBA
ABC], [U18133] 3-chlorobenzoic acid-3,4-dioxygenase;
[PCCBDABC] 2-chlorobenzoic acid-1,2-dioxygenase; [BS
U62430] 2,4-dinitrotoluene dioxygenase; [PSU4950
4] 2-nitrotoluene dioxygenase; [PPU24215] p-cumate-2,3-dioxygenase; [PSEPHT] phthalate-4
, 5-dioxygenase; [AB008831], [ACCANI], [D85
415] aniline 1,2-dioxygenase; [D90884] phenylpropionate 2,3-dioxygenase; [PPPOBAB] phenoxybenzoate dioxygenase; [AF060489], [AB001723] and [D890].
64] Carbazole dioxygenase.

Also useful are organisms whose genome contains genes encoding other dioxygenases, and these dioxygenases include: tetralin-
5,6-Dioxygenase, Sikkema et al., Appl. Eviron. Mi
crobiol. 59: 567-573, (1993); p-cumate-2,3.
-Dioxygenase, DeFrank et al. Bacteriol. 129: 1
356-1364 (1977); fluorenone 1,1
a-dioxygenase, Selifonov et al., Biochem. Biophy
s. Res. Comm. 193: 67-76 (1993); dibenzofuran-4.
, 4a dioxygenase, Trenz et al., J. Mol. Bacteriol. 176: 7
89-795 (1994); phthalate-3,4-dioxygenase, Eaton.
Et al., J. Bacteriol. 151: 48-58 (1982); and 2-chlorobenzoic acid-1,2-dioxygenase, Serifonov et al., Bioch.
emBiophys. Res. Comm. 213 (3): 759-767 (19.
95) etc. These and other dioxygenases suitable for use in constructing the enzyme libraries of the present invention are described in "DNA", filed Aug. 12, 1999.
SHUFFRING OF DIOXYGENASE GENES FOR P
Co-pending and commonly assigned US patent application Ser. No. 60 / 148,4 entitled "RODUTION OF INDUSTRIAL CHEMICALS"
50.

Once the hydroxyl group is introduced into the lead compound or other organic molecule,
Adding a functional group to this hydroxyl as described below (eg,
Glycosylation, acylation, etc.) is often desirable. Thus, the present invention also provides a library of organic molecule derivatives obtained by contacting these organic molecules with a first library of recombinant derivatizing enzymes, followed by a second library of recombinant derivatizing enzymes. And a method of contacting the library with. The enzymes of this second library are often (but not necessarily) those that catalyze the addition of chemical moieties to functional groups. Alternatively, the hydroxylated compound may be modified by chemical or other means known to those of skill in the art.

B. Halogenases Halogenases constitute another example of a class of derivatizing enzymes that can be used to obtain libraries of organic molecule derivatives. Halogenases generally halogenate aromatic rings, which can be part of complex natural or unnatural products, and other organic molecules of interest, for example, as lead compounds. Examples of suitable halogenases include: the halogenases PrnA, Prn.
B, PrnC (U74493; P. fluorescens), putative halogenases P1tM, P1tD, P1tA (AF081920; P. fluoresce).
ns), putative oxygenase / halogenase (Y16952; Amycolat
opsis orientalis). Although these particular enzymes have less than about 35% amino acid sequence identity, the polynucleotides encoding these enzymes are useful as probes to obtain more closely related halogenases that can be used for DNA shuffling. Is.

C. Other Substitutions Similarly, sulfur containing groups can be introduced into organic compounds. For example, thiols are commonly introduced to produce thiolate anions, which have a strong affinity for heavy metals. Often heavy metals are found in the enzyme active site. Derivatizing enzymes useful in these embodiments include, for example, the aryl sulfotransferase family. This family of enzymes can be used to transfer sulfo groups to the aromatic portion of organic molecules. The aryl sulfotransferase family has very high amino acid sequence identity (80%
, Etc., so that they can be easily shuffled together to produce a library of recombinant derivatizing enzymes. Examples of suitable sulfotransferase genes that can be used for recombination include, for example, the arylamine sulfotransferase (U33886; Homo sap
iens), phenol sulfotransferase (D85541; Macac
a fascicularis), phenol sulfotransferase (D2
9807; Canis familiaris), phenol sulfotransferase (U34753; Bos taurus), and minoxidil sulfotransferase (L19998; Rattus norvegicus).

In a further embodiment, one or more basic groups are replaced with existing functional groups. The most commonly used basic groups in medicinal chemistry are amines, amidines, guanidines and almost all nitrogen-containing heterocycles. Introduction of such a group into a molecule that already possesses biological activity has essentially the same solubilizing effect as the introduction of the acid functional group. Amines and basic heterocycles are substantially ubiquitous in effective drugs. For example, amines can be readily introduced by the use of acyl transferases or esterases with bifunctional compounds containing amines.

2. Addition of Chemical Moiety on Functional Group A further embodiment of the invention is one or more chemical moieties on a functional group present on the organic molecule of interest (eg, lead compound). Recombinant derivatizing enzymes and libraries thereof that can catalyze the addition of In these embodiments, the recombinant derivatizing enzyme of the invention is an enzyme capable of attaching a group on the functional drug portion of its core at a position that does not disrupt the function of the drug. Such conjugation may increase the solubility of the drug moiety (eg, as a prodrug).

The binding can be either reversible or irreversible. For reversible binding,
Examples include ester bonds, peptide bonds and glycosidic bonds. Irreversible bonds include, for example, bonds via O- and N-alkylation. The C—C bond can be prepared by grafting a side chain (eg, dimethylaminoethyl chain or morpholinoethyl side chain) or an acidic side chain (eg, carboxyl group, sulfone group, —OSO ₃ H, —PO ₃ H ₂ , —). OPO ₃ H ₂ ) or with a neutral group such as a glyceryl group. Larger solubilizing groups are also
It can be added using the enzymes and methods of the invention. Examples of these are -O-
_{CH 2 -CH 2 -COOH, -NH 2} -CH 2 -CH 2 -CH 2 -, - C = N-O-C
H ₂ -CO ₂ H, O-morpholinoethyl - and _{-O-CO-CH 2 -CH 2} -CO 2 but H include, but are not limited to.

Nonionizable side chains, including, for example, hydroxylated and polyoxymethyleneic side chains or various glycosides, are also included.
Can be combined to enhance solubility. This class of side chains also includes polyethylene glycol derivatives, which are also used for enhanced solubility and sustained release.

Examples of derivatizing enzymes useful for adding chemical moieties to existing functional groups on lead compounds or other organic molecules are glycosyltransferases, acyltransferases, amidases, N-methyltransferases, phosphotransferases, aryls. Such as sulfotransferase.

A. Acyltransferases Acylation is a type of modified chemistry that can theoretically provide a great deal of diversity in the derivatization of organic molecules. However, conventional chemical processes for acylation are typically non-selective and require multiple protection and deprotection steps. Enzymatic acylation in organic solvents by acyl transferases (including, for example, lipases and proteases) may provide certain advantages such as substrate selectivity, stereoselectivity and regiospecificity. However, it is unlikely that an acyl transferase can be obtained from a set of naturally occurring acyl transferases with various desired substrate, stereo and regiospecificities for any particular organic molecule. Thus, the present invention provides libraries containing a number of recombinant acyltransferases that can be used to synthesize lead compounds and acylated derivatives of other organic molecules.

Accordingly, the invention provides libraries of recombinant polynucleotides encoding lipase and protease enzymes, and acyltransferases. These methods include the steps of making a library of recombinant polynucleotides for use as a substrate polynucleotide that encodes an enzyme capable of performing an acylation reaction. Such enzymes include, for example, lipases and proteases. The reverse reaction of lipases and proteases in organic solvents can transfer various acyl groups onto the hydroxyl sites of complex natural products. These enzymes usually have broad substrate specificity but low activity.

The family of lipases can be readily identified, for example, from publicly available databases. An example of a family of lipases suitable for shuffling (greater than 50% amino acid identity) includes the following members: Y00557,
Vibrio cholerae; D50587, Pseudomonas s
p KFCC10818 (AAD22078), Pseudomonas ae
ruginose (BAA23128), P. aeruginosa (D505
87); Acinetobacter calcoacetius (AF047)
691); wisconsinensis (U88907 and 20
72017), Pseudomonas sp (P26877), Bacill
us subtilis (M74101); Bacillus pumilus
(A34992); Galactomyces geotrichium (A0
2813); Candida rugosa (WO99 / 14338); and Acinetobacter calcoaceticus (S61927).

Many genes encoding acyltransferases that use various carboxylic acid derivatives of coenzyme A as substrates are known, and the enzymes that catalyze these reactions are ubiquitous in prokaryotes and eukaryotes. Examples of nucleic acids suitable for use as substrates include, for example: galactoside 6-O acetyl transferase (EC 2.3.1.18); E. coli lacA (B0342
(LacA)) or lacA of another organism (GENBANK locus MG396
D02-orfl52 (lacA); MJ1064 (lacA), MJ167
8, MTH1067); serine O-acetyltransferase (EC 2.3)
． 1.30) (GENBANK locus B3607 (cysE), HI0606)
(CysE), HP1210 (cysE), SLR1348 (cysE)); alcohol O-acetyltransferase (EC 2.3.1.84) (for example, from Saccharomyces cerevisiae (locus YGR1
77C, YOR377W)); arylamine N-acetyltransferase (EC 2.3.1.118, as a typical GENBANK locus, Q0.
0267, D90786, Z92774, I78931, AF030398, A
F008204, AF042740); carnitine O-acetyltransferase (EC 2.3.1.7) (eg, of mammalian or yeast origin (GENBANK locus YAR035 (YAT1) and YM8054.01).
(CAT2)); Choline O-acetyltransferase (EC 2.3.1.
6) (eg of mammalian origin); and acetyl CoA: deacetylbindrin 4-O-acetyltransferase (EC 2.3.1.107) (St-P).
ierre et al., (1998) Plant J. 14: 703-713).

Suitable acyl donors for the improved enzymes of the present invention include, for example, acyl donor compounds that can act as donors for particular enzymes. Representative acyl donor substrates include vinyl esters, trifluoroethyl esters and other aliphatic esters, as well as benzyl and fatty acids. For example, Mozhaev et al. (1998) Tetrahedron 54: 3.
791-3982 (especially page 3976).

In a preferred form of the invention, the shuffled acyltransferase gene provides for transfer of acetyl groups and encodes an enzyme that uses an endogenous pool of acyl-CoA compounds in cells of the host microbial strain. The acyltransferase gene. The endogenous pool of acyl-CoA can also be enhanced by the introduction of acyl-CoA ligase (optionally improved by DNA shuffling) into the host microbial strain that undergoes the acylation reaction. Then, in this strain,
Exogenous acetate or other carboxylic acids in the medium are supplied, which are bound to CoA by acyl ligase. Suitable acyl ligases and methods for optimizing them are described in “DNA SH” filed on Aug. 12, 1999.
UFFRING OF MONOOXYGENASE GENES FOR P
Co-pending, commonly assigned US patent application Ser. No. 09 / 373,9 entitled "RODUTION OF INDUSTRIAL CHEMICALS"
28.

Compounds of interest for derivatization by acylation include, for example, natural products (eg, polyketides, flavonoids, peptide antibodies, etc.) and non-naturally occurring compounds. Such compounds find use, for example, as antibodies, chemotherapeutic agents and the like. Generally, the substrate molecule has one or more hydroxyl residues at which acylation can occur. Regioselectivity is especially important for molecules with multiple functional groups where acylation can occur. The method of the present invention provides a means by which an enzyme may be obtained that acylates a functional group of interest, but does not acylate other functional groups that may otherwise be susceptible to acylation.

Acylation of certain molecules can mitigate the detrimental properties. Among others, anti-cancer agents (including anti-cancer agents that act by disrupting the kinetics of microtubulin)
Compounds for which the method of the present invention is useful in developing derivatives of that drug with improved properties. Examples of these compounds include colchicine, colcemid, podoxophyllotoxin, taxol, vinblastine, vincristine, and the like. One particular example of a substrate of interest is epothilone, which is a potential anti-cancer drug candidate currently under investigation. Selective acylation of two hydroxyl groups on this compound can increase its water solubility. The recombinant acyltransferase libraries of the invention can be used to obtain derivatives that are specifically acylated at these positions. Further examples are rapamycin and FK506. Rapamycin C-28 hydroxyl group or FK506 non-dehydrated C-3
Acylation of the 5 hydroxyls can be used to separate their immunosuppressive activity from their neuroregenerative activity (Gold, BG (1997) Mol. Ne.
urobiol. 15: 285-306). It is known that the part of rapamycin or FK506 that binds to FKBP (FK binding protein) is responsible for nerve regeneration activity. Acylation is a function of FKB to effector protein (calculin)
It may disrupt the binding of P-rapamycin (or FK506). Therefore, acylation of the aforementioned hydroxyl groups destroys the calcineurin bond. Regioselectivity plays a major role in these modifications. This is because there are some hydroxyl groups in both molecules.

Screening libraries of recombinant polynucleotides encoding lipases, proteases or other acylating enzymes regardless of whether these polynucleotides are obtained by the DNA shuffling method as described above or other methods. Without using purified enzyme or partially purified enzyme or bacterial lysate or yeast lysate in an organic solvent system by one or more of the following screening methods:
Most easily done in vitro. For example, increased formation of naturally occurring products and small molecule acylated derivatives can be detected by detecting physical differences between the substrate and its derivative resulting from the enzyme-catalyzed reaction. These methods include HPLC
, Mass spectrometry, UV / Vis and IR spectroscopy, NMR and the like.

Another preferred method of the invention uses a labeled acyl donor precursor (eg, a labeled carboxylic acid or derivative thereof), which is a gene encoding a shuffled lipase, protease or other acyltransferase. Administered to cells expressing the library. The amount of label in the reaction product is measured. For hydrophobic reaction products, the derivative can be extracted into a suitable organic solvent,
Alternatively, a sufficient amount of hydrophobic porous resin beads (eg, XAD 1180, XAD-
2, XAD-4, XAD-8), solid phase extraction of these compounds may be used. In the case of radiolabeling, the scintillate dye can be present in the organic solvent, added to the sample, or chemically incorporated into the bead polymer. The latter constitutes a modification of the scintillation proximity assay.

As a method for detecting the regioselectivity of the acylation reaction, for example, HPLC,
And HTP mode include flow-through NMR spectroscopy. When NMR spectroscopy is used to determine the relative amounts of naturally occurring products or small molecule derivatives that have been acylated at different positions, the latter isotopes ( ¹³ C and / or ² H
) Obtained preferentially by the action of the enzyme on the labeled substrate. Another variation of the NMR technique involves the use of isotopically labeled precursors of acyl donor intermediates.

B. Glycosyltransferases Another example of a derivatizing enzyme of interest for making combinatorial libraries of organic molecule derivatives is glycosyltransferase. Glycosylation
It can increase bioavailability, reduce toxicity, and increase the water solubility of organic molecules (including lead compounds). Since glycosylation is difficult to chemically perform, novel sugars containing antibiotics (eg, novel glycopeptides and glycosylated macrolide antibiotics) are difficult to make.

However, the use of glycosyltransferases makes it possible to achieve glycosylation of organic acceptor compounds containing one or more hydroxyl groups. Therefore, due to the greater diversity in glycosylation capacity provided by the recombinant enzyme libraries of the present invention, many variants of organic molecules can be obtained. Using the techniques provided herein, novel enzymes are provided that can catalyze a variety of previously unobtainable glycosylation. For example, recombinant derivatizing enzymes in the libraries of the invention may exhibit altered specificity for acceptors (eg, complex natural and synthetic organic molecules) and donors (eg, different sugars). Increased ability to synthesize aminodeoxy sugars can also be obtained, for example, by biotransformation. Using the recombinant derivatizing enzymes of the present invention, new substrates can be accessed, new enzymatic activities can be generated and improved; different chemical processes can be replaced by biocatalysts, and large scale-up can be achieved. obtain.

Glycosyltransferases can be evolved using the methods of generating diversity described herein (including shuffling, for example) to produce recombinant glycosyls that exhibit optimal performance for a variety of different reaction parameters. Produces transferase. Representative reaction parameters include, but are not limited to, the specificity of the reaction, the degree of enzyme randomness and regiochemistry. For example, these enzymes transfer different nucleotide diphosphate (NDP) sugars and NDP sugar analogs; transfer sugars to different acceptor molecules; bind sugars at different positions as compared to naturally occurring enzymes. In order to have ambiguity for positions in the acceptor containing multiple sites and to catalyze multi-step glycosylation, it is optionally evolved.

In another embodiment, the enzyme can be evolved to produce a recombinant derivatizing enzyme that utilizes alternative sugars that are optionally synthetic. For example, activated sugars (eg desoxy sugars and sulphated sugars); unnatural sugars (eg nitrosylated sugars, sulphonated sugars, phosphonated sugars and didesoxy sugars).
r); polyalcohols such as inositol, inositol phosphates and inositol phosphonates; other sugar-like structures and compounds and alternative nucleotides.

Recombinant glycosyltransferases may also optionally replace sugars with alternative sugar receptors (polyketides, non-ribosomal peptides, complex molecules derived from organic synthesis,
And libraries of chemical compounds, including but not limited to). Other sugar receptors for the purposes of the present invention include aglycosyl vancomycin hydroc.
hloride) (peptide antibiotic), somatostatin (growth hormone), insulin and glucagon release inhibitor, cholic acid (surfactant steroid), nogaramycin (antitumor antibiotic), L-thyroxine (thyroid hormone),
Syringaldazine, aclarubicin (anti-tumor antibiotic and commercial RNA synthesis inhibitor), ritodrine hydrochloride (ritod)
line HCl (adrenergic agonists and smooth muscle relaxants), rifamycin (antibiotics), and ristomycin sulfate (antibiotics), but are not limited thereto. Each of these compounds is described by Chemweb (ht
tp: // www. chemweb. have a three-dimensional similarity to the vangomycin aglycone, as defined by the chemodynamic interface with the Available Chemical Database available via C / databases). These compounds and their intended sugar attachment points are shown in Figures 1-10. Other natural products of interest for glycosylation include, for example, lovastatin, aglycosylerythromycin, echinocandin (e
chinocandin), taxol and cephalexin.

Any molecule containing at least one hydroxyl group is optionally glycosylated with an evolved glycosyltransferase. Compounds of pharmacological interest are preferred. Sugar acceptors with more than one hydroxy group are optionally glycosylated at only one of their positions. Thus, different isomers can be produced by glycosylation at one of their positions or at another position. Alternatively, compounds with more than one hydroxy group are optionally glycosylated to different extents at different positions, for example when NDP sugars are limited. In yet another embodiment, the compound is a combination of an NDP sugar and a glycosyltransferase (providing repetitive glycosylation).
Is processed in multiple dimensions by.

[0110] In some embodiments, the glycosyltransferase is selected from glycosyltransferases that transfer hexose residues from UDP hexose derivatives. Preferred hexoses include, for example, D-glucose, D-galactose and DN-acetylglucosamine. Sugars of interest in conjugation using evolved glycosyltransferases include, but are not limited to, UDP-N-acetylgalactosamine, UD.
PN-acetylglucosamine, UDP-galactose, UDP-galacturonic acid, UDP-glucuronic acid, UDP-mannose, UDP-xylose, UDP
-Glucose, TDP-glucose, CDP-glucose, ADP-glucose, ADP-ribose, ADP-mannose, GDP-fucose, GDP-glucose, and GDP-mannose (all of which are Sigma (St, Louis,
s, MO)). Examples of deoxy sugars include: 2-deoxy-D-xylo-hexose, 2-deoxy-D-arabino-hexose, L-fucose, L-rhamnose, D-mycinose.
L-valalose, D-fucose, D-quinovose,
D-rhamnose, D-canarose, D-auriose (
D-oliose), D-digitose, D-boibinose, L-oleandrose (L-oleandr)
ose), chalcose, D-amisetose (D-ami)
cetose), L-rhodinose, ascarylose, abeqose, paratose, tiberose, colitose and the like. These and other sugars are described in Annu. Rev. Microbiol. 48,223-256 (1
994).

The present invention provides a method of obtaining a recombinant polynucleotide encoding a glycosyltransferase enzyme with enhanced specific properties, which polynucleotide is useful in the synthesis of glycosylated organic compounds. Increase sex.
In a preferred embodiment of the invention, a polynucleotide encoding the improved glycosyltransferase enzyme is introduced into a microorganism, which microorganism is added to the biocatalytic reaction mixture. In some embodiments, the glycosyltransferase is expressed by a species other than the microbial species from which the glycosyltransferase gene was obtained.

In a preferred embodiment of the invention, the glycosyltransferases used in the methods of the invention subject the nucleic acids encoding these enzymes to recombination and subsequent selection to produce enzymes with enhanced properties of interest. Optimized by identifying those recombinant polynucleotides that encode. For example, an enzyme that can be selectively glycosylated at only one hydroxyl group, can be regioselectively controlled to provide either of two possible isomeric compounds, or can be glycosylated on a broader range of compounds ( For example, enzymes that utilize various sugars and sugar analogs that are not normally utilized by naturally occurring glycosyltransferases, and enzymes that glycosylate various organic compounds to which naturally occurring glycosyltransferases cannot bind sugar molecules). , For those recombinant polynucleotides.

Subject to selection or screening to identify recombinant polynucleotides encoding recombinant glycosyltransferases with enhanced properties,
Libraries of recombinant polynucleotides include, for example, nucleic acid encoding these enzymes (ie, these nucleic acids are the substrates for recombination) and the various recombinant-based diversity described herein. It can be made by application of a method of producing sex, such as shuffling. Suitable sources of glycosyltransferase genes for use as substrates in the production of libraries of recombinant polynucleotides include, for example, A. orient
The gtf gene from alis is mentioned, which catalyzes the transfer of glucose to, for example, aglycosyl vancomycin. Enzymes that catalyze these reactions are ubiquitous in prokaryotes and eukaryotes.

One or more glycosyltransferases can be selected from the glycosyltransferase superfamily, aligned with similar homologous sequences, and shuffled to these homologous sequences. The glycosyl transfer reaction is
It is ubiquitous in nature and one of skill in the art can isolate such genes from various organisms using one or more of several known methods. The following shows examples of nucleic acids encoding glycosyltransferases that can be used as source nucleic acids for the production of recombinant libraries. The recombinant library is then screened to identify those that exhibit improved glycosylation of organic compounds (eg, altered substrate specificity). For example, inositol 1-α-galactosyltransferase, EC 2.4.1.123; phenol β-glucosyltransferase, EC 2.4.1.35 (NTU32643, NTU32644); flavone 7-O-β-glucosyltransferase, EC2.4. 1.81; flavonol 3-O-glucosyltransferase, EC 2.4.1.19 (A
B002818, ZMMCCBZ1, AF000372, AF028237, A
F078079, D85186, ZMMC2BZ1, VVUFGT); o-dihydroxycoumarin 7-O-glucosyltransferase, EC 2.4.1.1.
04; Vitexin β glucosyltransferase, EC 2.4.1.105;
Coniferyl alcohol glucosyltransferase, EC 2.4.1.11
1; monoterpenol β-glucosyltransferase, EC 2.4.1.12
7; arylamine glucosyltransferase, EC 2.4.1.71; s
n-glycerol-3-phosphate 1-galactosyl transferase, E
C2.4. 1.96; glucuronosyl transferase, EC 2.4.1.1
7 (RNUDPGTR, AA912188, AA932333); human UGT and isoenzyme (about 35 genes); salicyl alcohol glucosyltransferase, EC 2.4.1.172; 4-hydroxybenzoate 4-O-β.
-D-glucosyltransferase, EC 2.4.1.194; zeatin O-
β-D-glucosyltransferase, EC 2.4.1.203; D-fructose-2-glucosyltransferase, VFAUDPGFTA; and ecdysteroid UDP-glucosyltransferase (egt) MBU41.
All 999 can be used as substrates for the production of the recombinant libraries of the invention.

Additional suitable glycosyltransferase genes can be found in many microorganisms that one of ordinary skill in the art can isolate from various soil, sediment, air, and aqueous samples by enrichment culture techniques. Glycosyltransferases specifically isolated from soil bacteria glycosylate some polyketide aglycones, and such glycosylated natural products have many different biological activities (eg, antibacterial and antimicrobial properties). Cancerous). Genes encoding such enzymes are readily available from public databases. For example, glycosyltransferase (
S. antibioticus, AJ002638; Sac erythrae
a, Y14332; S.I. venezuelae, AF079762; peu
Cetius, L47164 and S. fradiae, X81885). These genes share greater than 50% amino acid sequence identity, and any two or more are therefore ideal for shuffling together as a family.

As an example, the glycosyltransferases used for the initial shuffling were gtf from different Amycolatopsis orientalis strains.
A, gtfB, gtfC, gtfD, and gtfE. These genes encode glycosyltransferases that transfer the sugar moiety to the aglycones of vancomycin and eremomycin, which are non-ribosomal peptide antibiotics. Zmijewski & Briggs FEMS Microbiolo
gy Letters 59, 129-134 (1989). Solenber
g et al., Chem & Biol. 4, 195-202 (1997). Wagenin
gen et al., Chem. & Biol. 5,155-162 (1998). For example,
GtfB and gtfE transfer glucose from TDP-glucose or UDP-glucose to vancomycin. The glycosyltransferase gene is
Share the similarity between 59% (gtfA-gtfD) and 82% (gtfB-gtfE). The protein sequences are 52% (gtfA-gtfD) and 80% (gt
fB-gtfE). The five published genes are different Amycolatopsis orientalis ssp orients.
alis strains (gtfD and gtfE from ATCC 43490 or ATCC 43491, and gtfA, gtfB, gtf from NNRL18098).
Can be amplified from C). Even more numerous, but uncharacterized, glycosyltransferase genes of interest have been described in other A. orientali
s strains (eg, ATCC 19795, 21425, 35164, 15165, 1,
5166, 39444, 43333, 53550, and 53630) to PC
R-amplified and cloned into appropriate cloning and expression vectors. An additional gene is Amyc, a balhymycin producer.
olatopsis mediterranei DSM5908 (Pelze
r et al. (1997) J. Am. Biotechnol. 57: 115-128), and other Amycolatopsis strains. E. coli of the protein encoded by gtf. Expression in E. coli can be tested by either SDS-PAGE, Coomassie blue staining, and / or by Western blot if a detection tag was added. For example, single clones of gtfB and gtfE can be tested for their wild type activity. For example, gtfB
And gtfE transfer from TDP or UDP glucose to the aglycone of vancomycin. Folena-Wassermann et al. of A
ntibiotics 39, 1395-1406 (1986). In vitro glucosylation of vancomycin aglycone can be monitored by reverse phase HPLC. Solenberg, et al., Chem. & Biol. 4,195-202
(1997). Subsequently, functional gtfB and gtfE clones, and other genes expressing polypeptide chains of the desired size (eg, gtfA, g
Several clones of tfC, gtfD, etc.) are used to generate PCR products of the gtf gene in the context of screening vectors. DNAs of each PCR product
The eI fragment is generated and reconstructed by various shuffling methods as described above, for example. Typically, fragment sizes are 25 base pairs and 25
Although between 0 base pairs, this size is easily determined experimentally by methods well known in the art.

C. Methyltransferase Methyltransferase is another example of a derivative enzyme of interest that can add a chemical moiety present in a lead compound or other organic molecule to a functional group. S-adenosylmethionine (SAM) -dependent methyltransferases (MTs) are, for example, proteins, nucleic acids, sugars, polysaccharides, lipids, lignins, and methyl ester derivatives of various low molecular weight compounds (eg macrolides), methyl. It constitutes a class of enzymes that form ether derivatives, methylthioether derivatives, methylamine derivatives and methylamide derivatives. SAMs have activated methyl groups that are efficiently transferred to nucleophiles with a wide range of chemical reactivity. The transfer of activated methyl groups from the SAM to the recipient nucleophile is thermodynamically favorable and therefore drives the methyl transfer reaction to essentially completion.

One class of methyltransferases of interest is N-methyltransferases. By way of example, the following N-methyltransferases have at least 59% amino acid sequence identity and thus constitute a family that is particularly well suited for shuffling (putative TDP-N-dimethyldesosamine-N-).
Methyltransferase (U77459; Saccharomyces er
ythreaa), methyltransferase (AJ002638; S. ant)
ibioticus), N, N-dimethyltransferase (AF07976)
2; S. venezuelae), N-methyltransferase (X8188)
5; S. fradiae). This family of enzymes usually methylates the amine groups of aminodeoxy sugars that are added to complex natural products.

Also of interest are O-methyltransferases, several families of which are known. For example, the following family of methyltransferases can methylate the hydroxyl groups of complex natural products: 31-demethyl-FK506 methyltransferase (U65940; Streptom).
yces sp), methyltransferase (X86780; Strepto
myces hygroscopicus), carbomycin 4-O-methyltransferase (D30759; Streptomyces thermotte)
olerans), and O-methyltransferase (M93958; St
reptomyces mycarofaciens). These family members have greater than 45% identity at the amino acid level.

D. Amidase The present invention also provides a recombinant library of amidases. This family of enzymes can be used to introduce amide groups into organic molecules. The reverse of the amidase reaction converts the carboxylic acid group to a carboxylic acid amide. One such family suitable for use in the methods of the invention includes the following amidases that are at least 55% identical at the amino acid level: N-acetyl-anhydromuramyl-L-alanine amidase. (AF082575; Pseudomo
nas aeruginosa), N-acetyl-anhydromuramyl-L-alanine amidase (U40785; Enterobacter cloacae).
), AmpD protein (X15237; E. coli), and AmpD protein (U32716; Haemophilus influenzae Rd.
).

E. Phosphotransferases Addition of phosphate groups to existing functional groups of lead compounds or other organic molecules is also of interest. Thus, the present invention provides a library of recombinant phosphotransferases that are useful for obtaining phosphorylated organic molecule derivatives. As an example, the macrolide and peptide phosphotransferase families, whose members have at least 36% amino acid sequence identity, can be subjected to recombination (eg, macrolide 2'-phosphotransferase I (D162).
51; E. E. coli), macrolide 2'-phosphotransferase II (D
85892; coli), viomycin phosphotransferase (X02
393; Vinaceus)). This group of enzymes transfers phosphate groups onto macrolides or peptide antibiotics to inactivate them. By using a library of recombinant phosphotransferases, one of skill in the art can
It is possible to phosphorylate different sites of macrolides or peptide antibiotics.

F. Other Enzyme Classes Enzyme classes other than those described above are also very important for introducing or modifying functional groups in lead generation and / or optimization. For example, enzymes capable of catalyzing redox reactions are important for oxidizing functional alcohols to aldehydes / ketones or for reducing aldehyde / ketone groups to alcohols in organic compounds. These newly created groups can then be further modified by other classes of enzymes as described. One such family suitable for shuffling is the family of lactate dehydrogenases that convert ketones to alcohols with amino acid sequence identities greater than 80%: (Y00711, Homo sapiens; U07181, Ra.
tus norvegicus; 77022A, Sus scrofa do
(Mestica; L79954, Tranchemys script, etc.)
. Alcohol dehydrogenases are another family of enzymes that oxidize alcohol groups to aldehydes. Suitable genes for this enzyme family are readily available for shuffling (M88409, Homo sapiens; L
15704, Peromyscus maniculatus; 156882,
Struthio camelus; P80222, Alligator mi
ssissippiensis). Shuffling of these two families of enzymes can alter their substrate specificity for more complex organic compounds.

An enzyme capable of oxidizing a sulfide to a sulfoxide and a thiol to a thioaldehyde,
And other enzyme families such as enzymes capable of catalyzing cyanohydrin formation and epoxidation etc. are also targets for DNA shuffling and are therefore valuable catalysts for use in combinatorial biosynthesis.

C. Use of Recombinant Derivatizing Enzyme Library to Obtain Recombinant Library of Organic Molecule Derivatives The present invention provides, in a further embodiment, a method for obtaining a library of organic molecule derivatives. To do. These methods include the step of contacting an organic molecule (substrate) with a recombinant derivatizing enzyme and other necessary reactant libraries to form a library of organic molecule derivatives. The derivatizing enzyme described above catalyzes reactions such as: a) modification of one or more functional groups present on an organic molecule; b) onto one or more functional groups present on an organic molecule. Addition of chemical moieties; or c) introduction of new functional groups onto the organic molecule.

(1. Organic molecule of interest for derivatization) Examples of the organic molecule of interest include those having a pharmacological activity, a herbicide activity, or an insecticide activity. Some organic molecules of interest include natural products (eg,
There are antibiotics (including, for example, polyketide antibiotics, steroid antibiotics, non-ribosomal peptide antibiotics, etc.). For example, steroids are a very widely used basic structure for drugs, whereby substituents on the ring target the drug to many different therapeutic targets. Most of these are from natural sources and are screened for efficacy. Substituents observed on steroid drugs include hydroxyl groups, methoxy groups, alkoxy groups, glycosylations, sulphations, halogenations, double and triple bonds, carbonyls, and the like. Chemical derivatization of the steroid cyclic structure can be accomplished at several well-described sites or by modification of naturally occurring structures or by non-naturally occurring derivatives thereof.
Can be easily achieved.

Cyclic glycopeptides and macrolides such as vancomycin and erythromycin are also chemically distinct structures that can be modified by applying shuffled enzyme libraries. There are many such structures isolated from nature, and described in the literature and in the company's storerooms, which are:
It has interesting biological activity but is otherwise lacking. Toxicity, bioavailability, solubility, pharmacokinetics, lack of selectivity are some of the reasons why drug candidates are unable to become drugs. Application of shuffled libraries can be used to improve these and other features.

Prostaglandins, alkaloids, anthraquinones are another family of molecules that have many biologically active members. These are also good candidates for improvement with shuffled enzyme libraries.

Specific examples of pharmaceutical compounds that can be derivatized using recombinant derivatizing enzymes include, for example, tuboclarin chloride, alcuronium chloride, pancuronium bromide, vecuronium bromide, atracurium besylate, 776C.
85,7 CIMe-MDO-CPT, 9-aminocaptothecin, A-007, A
-108835, A-121798, pullurea glycosides A and B, lanatosides A, B and C, α-acetyldigoxin, β-acetyldigoxin, digoxin, β-methyldigoxin, k-strophanthoside, k-strophanthin-β. , Convaloside, convalatoxin, glucosilaren A, psilalen A, procillaridin, psiralenin. Cholekines and cholekines
Tic) drugs (including, for example, hymecromone, febupol, chenodeoxycholic acid, and ursodeoxycholic acid) are also of interest. Fluocortron, parametricsone, dexamethasone, betamethasone, cortisone, hydrocortisone, prednisone, prednisolone, triamcicolone acetonide, triamcinolone, methylprednisolone, and prednilidene are suitable glucocorticoids for derivatization. Corticoids of interest also include, for example, predonic carbate, hydrocortisone aceponate, fluocortin butyl, ioteprednol etabonate, and the like.

2. Enzymatic Reaction To obtain a library of organic molecule derivatives, its substrate is contacted with a member of a library of recombinant enzymes. The enzymatic reaction can be carried out in a number of ways including, for example, the use of whole cell biotransformation, permeabilized cells, cell lysates, and purified proteins.

Whole cell biotransformation occurs when its substrate (eg, an organic molecule) is exposed to cells containing a library of recombinant derivatizing enzymes. The library can be expressed as a surface protein on a replicable genetic package (eg, phage or yeast display) or as a secreted protein that interacts with the substrate in solution. The enzyme can also be expressed inside the cell, where the substrate diffuses into the cell before the reaction takes place. In each case, the product resulting from the derivatization of the enzymatic activity is isolated from the cells by methods known to those skilled in the art including, for example, centrifugation, precipitation, extraction with organic solvents, and filtration.

Cells expressing the library can be permeabilized by the addition of a number of well known permeabilizing agents such as polymyxin B sulfate. The level of permeabilizing agent can be modified so that the substrate and product are free to diffuse through to the enzymes of the library and again out of the cell. At higher levels of permeabilizing agent, the protein can be released in solution. The compound of interest is isolated on whole cells.

The library can be used as a cell lysate, whereby cells expressing the library are disrupted by the addition of detergent, PMBS and lysozyme or the addition of well known lysis conditions including sonication. To be done. Cell debris may, but need not, be removed prior to the reaction by centrifugation. The substrate is then added to the lysate after incubation at a defined temperature for a defined length of time.
The product is then extracted as before and analyzed as described below.

Alternatively, the recombinant derivatizing enzyme encoded by the library can be purified by a number of well-known techniques before being used to screen or make derivatives of organic molecules. Such methods include, for example, gel filtration, to produce either partially or fully purified protein,
Ion exchange, affinity chromatography, or hydrophobic chromatography is included. Many other purification methods are known to those of skill in the art. The purified protein is then exposed to the substrate under conditions that favor enzymatic activity.

The reaction conditions used for the conversion are optimized by standard methods, including the use of optimal salt levels, buffers, temperatures, and reaction lengths, for maximum enzyme turnover. . The substrate and other substrates consumed in the enzymatic reaction are preferably used in concentrations that promote high turnover rates.

Contacting organic molecules and other reactants with a recombinant derivatizing enzyme
This can be done by using a complete library of enzymes simultaneously, or by using pools of recombinant enzymes from the library or a single recombinant enzyme in each reaction. If a pool is used, that pool can be deconvoluted to isolate particular clones that exhibit the desired activity once the active pool is identified using the methods described. For example, colonies expressing each member of the library of recombinant derivatizing enzymes can be placed in microtiter plates or other suitable vessels and subjected to high throughput screening.

In some embodiments, members of a library of recombinant enzymes are immobilized on a solid support prior to being contacted with other reactants. For example, a recombinant polynucleotide encoding an enzyme can be introduced into an expression vector that also contains the coding sequence for the tag, so that the recombinant derivatizing enzyme is expressed as a fusion protein with a tag. Alternatively, the tag can be linked to a derivatizing enzyme after its expression. The tag is typically readily available to the corresponding member,
And it is a member of a binding pair that can be immobilized on a solid support. For example, the recombinant enzyme can be expressed as a fusion with biotin, which can then be immobilized by binding to streptavidin. Other suitable binding pairs include, for example, maltose binding protein and amylose, histidine tags, and immobilized metal ions,
Glutathione-S-transferase, and reduced glutathione, streptavidin binding and streptavidin, epitope tags (eg E-.
Tag, myc-tag, HAG-tag, His-tag) and the corresponding antibodies, chitin-binding domain and chitin, S-tag and RNase minus S-peptide variants, cellulose-binding proteins and domains and cellulose, thioredoxin and DsbA and A thiol compound (eg Thibond ^™ ),
Polycation tags (eg polyarginine) and polyanion columns, Ig
G and IgG derivatized peptides and proteins A, protein G, etc., including calmodulin-binding peptides and calmodulin, histactophilin and immobilized metal chelate chromatography.

The members of the binding pair to which the tag bound to the enzyme binds are preferably bound to a solid support. Suitable solid phase supports for use are known to those skilled in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement. Exemplary solid supports include glass, plastics, polymers,
Metals, non-metals, ceramics, organic substances, etc. are included. The solid support can be flat or planar, or can have substantially different conformations. For example, the substrate can be particles, beads, chains, sediments, gels, sheets, tubes, spheres, containers,
It can be present as a capillary, pad, section, film, plate, dipstick, slide, etc. Magnetic beads or particles such as magnetic latex beads and iron oxide particles are examples of solid substrates that can be used in the methods of the invention. Magnetic particles are described, for example, in US Pat. No. 4,672,040 and are described, for example, in PerSeptive Biosystems, Inc. (Fram
ingham MA), Ciba Corning (Medfield MA)
, Bangs Laboratories (Carmel IN), and Bi
oQuest, Inc. (Atkinson NH). The substrate is chosen to maximize the signal to noise ratio, primarily to minimize background binding, and for ease of washing and cost.

Separation of recombinant enzymes from other cellular components, or from reactants, etc. may be accomplished by emptying a reservoir, such as a microtiter plate well, by removing beads or dipsticks from the reservoir, or Diluting can be done by rinsing the beads (beads with iron nuclei can be easily isolated and washed using magnetism), particles, chromatography columns or filters with wash solution or solvent. . Separation steps sometimes include an extended rinse or wash or multiple rinses or washes. For example, if the solid phase substrate is a microtiter plate, the wells typically interfere with subsequent screening of organic molecule derivatives (eg, salts, buffers, detergents, non-specific proteins, etc.). It may be washed several times with a wash solution containing the components of the resulting reaction mixture.

The libraries of recombinant derivatizing enzymes provided by the present invention are not only useful for obtaining libraries of organic molecule derivatives, but those skilled in the art will appreciate that recombinant catalyzed catalysis of a particular reaction of interest. It is also useful to provide a source from which the enzyme can be identified.
For example, once a particular organic molecule derivative is identified as having the desired properties, one of skill in the art can identify particular recombinant enzymes from enzyme libraries that can catalyze the formation of particular derivatives.

3. Screening of Organic Molecular Derivatives Recombinant derivatizing enzyme libraries are useful for the production of recombinant libraries of organic molecular derivatives. The organic molecule derivative is then screened to identify those that exhibit the desired activity. In these embodiments, the products of the screen are often compounds not previously made. further,
Recombinant enzyme libraries provide a source by which one of skill in the art can identify enzymes that catalyze certain known modifications of organic molecules. Thus, for example, one of skill in the art can obtain from a library enzymes that allow enzymatic synthesis of known compounds that were previously only synthesized by less efficient methods (eg, chemical synthesis). it can.

Once a library of organic molecule derivatives has been synthesized, the library is
In general, they are screened to identify derivatives of particular interest. In general, to identify derivatives that show an improvement in a particular biological activity, one of skill in the art can use bioassays designed to allow detection and / or quantification of the desired activity. Desired biological activity (including cytotoxicity, genotoxicity, etc.), desired bioavailability (including properties such as plasma half-life, renal clearance), desired physicochemical properties (water solubility, lipid solubility) , Solubility in organic solvents (eg, n-octane), water solubility, pH stability (eg, low pH environment of stomach), temperature stability, resistance to gastrointestinal enzymes, resistance to liver enzymes, resistance to plasma enzymes , Tissue permeability (eg, dermis, mucosa, etc.), blood-brain barrier permeability, as well as other desired properties that can be achieved by derivatization, are all screened for Can be done intentionally,
Alternatively, an analysis of the structure of compounds in the library to identify those with a particular structure of interest can be performed first. Once a compound having the desired biological activity is identified, structural analysis can be utilized to identify structural features conferred by the library of recombinant derivatizing enzymes.

In most cases, the recombinant derivatizing enzyme present in the library will be expected to chemically modify a given substrate in a predictable manner. For example, glucosyltransferases transfer the sugar moiety of a substrate onto an amine or hydroxy. This leads to predictable changes in the physical behavior of the molecule, which can be used for screening. The chemical transformations catalyzed by a particular library on any given substrate tend to be the same. For example, glycosyltransferases place sugars on substrates, methyltransferases tend to add methyl groups, and P450s tend to add hydroxyl groups and the like.
This allows general screening methods to be devised for each library. For example, glucosyltransferases always produce sugar substrate linkages. Thus, certain chemical tests on linked sugars detect product formation. Kinase libraries transfer phosphate groups to substrates, and specific phosphate tests detect the presence of product.

A number of analytical screening tools are available to determine the structure of compounds in combinatorial libraries. For example, numerous methods are known that are capable of detecting low concentrations of compounds in high throughput formats, including flow analysis NMR and mass spectrometry. These analysis tools or UV
Other analytical tools including / Vis and IR spectroscopy, fluorescence spectroscopy, luminescence, etc. can be used both for detecting and quantifying new compounds produced in enzymatic reactions.

A 100 percent turnover for the product of the substrate is not expected in the library screen, and so the analytical technique is preferably set up to detect the specific alteration produced by the enzymatic activity. . For example, the presence of an enzyme having a methyltransferase activity for a particular substrate of interest in a library of recombinant enzymes may result in 14am in mass spectrometry after contact with the enzyme.
It can be detected by observation of an increase in u. Therefore, the changes in the chemical structure of the substrate caused by the library can often be specifically monitored and detected. These can then be correlated to members of a library of recombinant enzymes that catalyzed a particular reaction.

Another approach to detect the presence of a recombinant enzyme that catalyzes a particular reaction on a new substrate in an enzyme library is, for example, the incorporation of molecular markers during the course of the reaction. Suitable labels include, for example, radiolabels (eg, ³ H
, ¹⁴ C, ³² P, etc.) are included. This can be accomplished using a radioactive co-substrate such as ³ H ₃ methyl S adenosylmethionine, which only labels the methylated product of the reaction. Other labels may also be used; many are known in the art. For example, glycosylation can be detected by the use of sugar moieties that include a label.

In certain cases, the product of action of the shuffled library on the substrate provides a more stable product than the substrate against external stresses such as extreme pH, increased solubility of the compound in certain solvents. Expected to do. This change in observation can also be monitored by an appropriate analytical or bioassay method.

In some cases, detection of the newly formed product may require separation of the product from its substrate by standard chromatographic methods (eg TLC, HPLC, CE, or GC). . After this, spectroscopic or other methods (
Flame ionization, mass spectrometry, for example) is performed to detect the formation of novel compounds of interest.

EXAMPLES The following examples are provided to illustrate the present invention, but not to limit the present invention.

Example 1 Generation of Glycosyltransferase Enzyme Library and High-Throughput Screening for Desvancosamine Vancomycin Production This example can generate a library of recombinant glycosyltransferases and desvancosamine A method is described in which the enzyme for the production of vancomycin can be used.

(A. Amycolatopsis orientalis ssp ori
Cloning of gtfA, B, C, D and E genes from S. entalis strain) (1. Generation of DNA encoding gtf) (Preparation of genomic DNA) Amycolatopsis orientalis ssp. orien
talis strains ATCC 43490 and NRRL 18098 are obtained from ATCC and NRRL. Starter cultures on agarose petri dishes are prepared according to the supplier's recommendations. Liquid cultures are grown in TBS at 25 ° C-28 ° C for 2-5 days. The genomic DNA was subjected to standard procedures (Ausubel et al. (1987)
) Current Protocols in Molecular Biol
Ogy, 1st edition, John Wiley & Sons, Inc. , NY).

PCR with add-on primers PCR was performed using the following, using the enzyme supplier (New England Bi).
olabs) and 1.5 M betaine and 1-3.5 mM MgS
Performed in a volume of 50 μl in the presence of O ₄ : genomic DNA, 1 pmol of gene-specific primer, 200 μM dNTP, 2 units of Deep Vent Po.
Lyumerase and 0.2 units of Deep Vent Polymer
5'-3 'exonuclease activity deletion mutant of ase. In all cases a hot start with wax beads (MβP) is used. In the DNA engine thermal cycler, set the cycle to the following scheme:
5 cycles of 95 ° C for 5 minutes; 95 ° C for 45 seconds, 76 ° C for 1 minute and 20 seconds for 5 cycles;
5 cycles of 45 ° C. for 45 minutes, 1 minute 20 seconds at 75 ° C .; 5 cycles of 45 seconds at 95 ° C., 1 minute 20 seconds at 74 ° C .; 10 cycles of 45 seconds at 95 ° C., 1 minute 20 seconds at 73 ° C. 9
10 cycles of 45 seconds at 5 ° C and 1 minute 20 seconds at 73 ° C. All primers are designed according to sequence entries U84349 and U84350. For amplification of gtfA, the primer gtfA. For and gtfA. Use Rev.
For amplification of gtfB, the primer gtfB. For and gtfB. Rev
To use. For amplification of gtfC, the primer gtfC. For and gt
fC. Use Rev. For amplification of gtfD, the primer gtfD. Fo
r and gtfD. Use Rev. For amplification of gtfE, primer g
tfE. For and gtfE. Rev is used (Table 1).

[0152]   (Table 1)   (Primers, oligonucleotides, polynucleotides)

[0153]

[Table 9] The resulting PCR product is digested with NdeI and EcoRV. The digested PCR product corresponding to the gft gene was subjected to agarose gel electrophoresis and QIAEXI.
Purify by I (Qiagen).

2. Properties and Structure of Vector pCKZEBB Vector pCKZEBB was added to pAK400 (Krebber et al. (1997) J).
． Immunol. Meth. 201: 35-55. pAK400
The following features of are maintained. The lacI ^q gene is maintained for repression of the lac operon. Maintaining a transcription terminator (hp ^t ) between the lacI ^q gene and the lac promoter (lac ^{p / o} ) to terminate non-induction by terminating the read through transcription from the lacI promoter to the operon controlled by the lacI promoter. Reduces sexual basal expression. The lac promoter operator was maintained for transcription initiation and transcriptional control. T7 before the target gene start codon
The T7g10 leader from phage gene 10 was maintained to allow strong translation initiation from the ATG start codon at the NdeI restriction site. NdeI-Hind
Behind the expression cassette controlled by the lac promoter operator of III,
The lpp transcription terminator (lppt) is encoded, followed by the f1 origin of replication that enables single-stranded DNA production, followed by the chloramphenicol resistance gene (cam ^R ), and double-stranded DNA replication. There is a ColE1 origin for

In pCKZEBB, the lac promoter operator-controlled polycistronic message replaces the lac promoter operator-controlled monocistronic message in pAK400. The operon transcribed in the lac promoter is the only NdeI and H of the pAK400 vector.
It is located between indIII. In pCKZEBB, a variant of the lacZ gene (the start codon ATG was integrated at the NdeI site, the internal NdeI was removed, an EcoRV site was added to the end of the gene before the stop codon, and the inverted EcoRV lacZ piece was inserted into the vector. Resulting) is inserted as a stuffer fragment into the NdeI EcoRV target gene cloning site. This lac
The Z fragment is replaced by the target glycosyltransferase gene.
After lacZ, a biotinylated tag is encoded (amino acid sequence), followed by t
There is a translational coupling tag derived from the end of rpB. Both tags are fused in-frame to the target glycosyltransferase when it replaces the lacZ stuffer fragment. The A nucleotide of the stop codon (TGA) of the translation coupling tag is a green fluorescent protein-encoding gene (GFP; Crameri et al.
Biotechnol. 14: 315-319) and constitutes a part of the translation initiation codon. This GFP gene is followed by the PCR-cloned birA gene containing the ribosome binding site from BL21 (DE3). There appears to be sequence ambiguity in this birA gene. This is because there is an NcoI restriction site in this region. A map of pCKZEBB is shown in Figure 19 and the nucleotide sequence of this vector is given as SEQ ID NO: 19.

E. coli transformed with pCKZEBB. E. coli does not become green fluorescent when grown on 30 μg / ml chloramphenicol and 1 mM IPTG. Replacement of the stuffer lacZ fragment with the biotinylated translational coupling tag in frame with the full length target gene results in: A) IPTG of the target gene
Inducible expression renders plasmid-harvesting bacteria green fluorescent by translational coupling to the GFT gene (Openpenheim and Yanofsky (1980) G
enetics 95, 785-795), and B) the target gene is birA.
Derived biotin holoenzyme ligase (Smith et al. (1998) Nucl. Aci
ds Res. 26: 1414-1420) via a biotinylated tag (Sch
atz (1993) Bio / Technology 11: 1138 to 1143.
) Is biotinylated in vivo.

3. Cloning of gftPCR into pCKZEBB The vector pCKZEBB is cut with NdeI and EcoRV to remove the lacZ gene stuffer in two parts. The resulting vector is dephosphorylated by using calf intestinal phosphatase. D corresponding to this vector
The NA fragment is isolated from the agarose gel by QIAEXII (Qiagen). The PCR product digested with EcoRV and NdeI described above is ligated into this vector fragment according to standard procedures. After ligation, E. coli
TG1 electrocompetent cells (Stratagene) were electroporated with this ligation and plated on LB-Agar plates containing 30 μg / ml chloramphenicol and 1 mM IPTG and plated at 37 ° C. Grow overnight. Green fluorescent colonies showing varying degrees of fluorescence are selected and plasmid DNA is prepared.

4. Restriction Analysis and Sequencing The resulting vector is analyzed with restriction enzymes and the clones containing the insert are sequenced. A plasmid is identified that expresses one of the glycosyltransferases as a biotinylated-translational coupling tag fusion protein. Clones carrying the gene corresponding to the published sequence are used as shuffling templates.

(B. Family shuffling, single, dual (do)
recombination and mutation of gene combinations of triple, triple, quadruple and all 5) 1. Amplification of wt gene in pCK vector Glycosyltransferase gene (flanking region from some vectors) From the resulting plasmid using the polymerase according to the manufacturer's recommendations and using the primers CK. For3 and H3. Amplify by Rev. The PCR is purified on a Qiaquick column (Qiagen).

(2. Generation of Random DNA Fragment) A PCR product derived from any of these plasmids was used as DNAseI (Boehh).
digest with a ringer). The reaction is stopped on dry ice and fragments of the desired size range are isolated from a 2% agarose gel using glass filter disks (Whatman) and dialysis membranes (Spectrapor) (Stemmer (1994) Proc. Nat'l. Acad. Sci.
USA 91: 10747-10751 and Stemmer (1994) Na.
true 370: 389-391).

3. Glycosyltransferase Gene Assembly Several concentrations and ratios of DNAs for each family assembly reaction.
Adjust the e-digested DNA fragment, as well as the PCR cycling parameters to obtain the maximum amount of shuffled gene in step 4 (Cr
ameri et al. (1998) Nature 391: 288-291, Chris.
tians et al. (1999) Nature Biotechnol. 17: 259
~ 64).

4. Rescue of Glycosyltransferase Gene by PCR 2 μl of final assembly reaction is used as template. Final PCR
In the reaction, 1 μM of primer CK. For2 and N3. Rev, 0.2
Add each unit of mM, 1 unit of Tag polymerase. The following PCR
Set parameters: 96 ° C for 3 minutes for 1 cycle; 96 ° C for 0.5 minutes,
30 cycles of 0.5 minutes at 60 ° C, 1.5 minutes at 72 ° C; 1 minute at 72 ° C for 5 minutes
cycle.

C. Cloning of the gtf PCR product into pCKZEBB The expression vector pCKZEBB and the PCR rescued shuffled glycosyltransferase gene are digested with XbaI and EcoRV. The vector pCKZEBB is further dephosphorylated. This vector fragment and the PCR fragment encoding the glycosyltransferase are isolated from an agarose gel and ligated together.

Electrocompetent E. E. coli TG1 was transformed with this ligation mixture and shaken for 1 hour at 37 ° C. before plating on LB agar containing 30 μg / ml chloramphenicol, 1% glucose and overnight at 37 ° C. Proliferate.

D. Glycosyltransferase Library Prescreening, Masterplate Generation, and Expression Colonies are picked in LB-Cam-Glucose and grown overnight at 37 ° C. to generate master plates. From the master plate, LB-Cam-
Align the colonies on IPTG-Agar and incubate the plate at 37 ° C overnight. Green fluorescent colonies are identified by exposing the plate to 365 nm UV light. Each green fluorescent colony from the master plate is realigned in a 96 well plate filled with 100 μl of 2YT-Cam 30-1% glucose in each well and grown overnight at 37 ° C.
Transfer 50 μl culture to 1 ml 2YT-Cam30-1 mg / ml biotin,
And it is made to grow at 16 degreeC for 7 hours. 50 μl of 100 μM IPTG is then added and the culture is grown overnight at 16 ° C.

E. Lysis of Cells with a Combination of Lysozyme and Polymyxin B Sulfate The culture is centrifuged (15 minutes at 4000 rpm) to pellet the cells. 500 μl of this cell pellet containing 50 mM ammonium formate (pH 7.4)
Wash with and pellet again. These cells were treated with 300 μl of lysis buffer (10 μl in 10 ml of 1 mg / ml polymyxin sulfate B (Sigma)).
L's Ready to Lyse lysozyme (Epicentre),
2 μL of RNAse A (Qiagen), 2 μL of DNAse I (Boehr
inger), 2 μL of 1M MgSO ₄ , 50 mM ammonium formate (pH
Resuspend in 2 mM DTT in 7.4) and stir at ambient temperature for 30 minutes. The lysate is then clarified by centrifugation (4000 rpm for 15 minutes).

F. Purification of Proteins from Single Colonies by Magnetic Beads Streptavidin-coated magnetic beads are aligned in 96 well plates. The beads are washed with buffer (50 μM ammonium formate (pH 7.4), 2 mM DTT) using the magnetic properties of the beads and resuspended in 20 μl buffer per well. Clarified cell lysate (100 μL) is transferred from the lysis plate to the beads and incubated at ambient temperature for 15 minutes. The beads are then washed 5 times with buffer (150 μL) and finally resuspended in 20 μl buffer.

G. Performing In Vitro Modification of Compounds with Glycosyltransferases from the Library The reaction mixture (80 μL) is added to the purified protein on the beads and the beads are stirred overnight at ambient temperature. The reaction mixture contained 150 μM vancomycin aglycone (J. Chem. Soc. Chem ,. Commun. (1988) 1
500 μM UDP glucose, 2 mM DTT in 50 mM ammonium formate, pH 7.4.
The reaction is quenched by the addition of 1 volume of methanol, and the mixture is centrifuged (2000 rpm for 5 minutes). The supernatant (100 μL) is transferred to a new 96-well plate and subjected to mass spectrometry.

H. Measurement of Glycosylation Occurrence The quenched reaction mixture (10 μl) is injected into a triple quadrupole electrospray mass spectrometer set in positive mode. The molecular ion was passed through a first quadrupole (1143 amu for vancomycin aglycone, 1305 amu for desvancosamine vancomycin) and subjected to collisions in a second quadrupole, then 100 amu at the third quadrupole. The peak detection of the daughter ion of. The integral of the peak obtained from this process is directly proportional to product formation. This determines the relative fitness of library clones in the product of desvancosamine vancomycin.

I. Repeated Use of This Procedure These steps can be repeated if desired. For example, steps B-H can be performed using multiple genes encoding variants of a particular derivatizing enzyme, using a single gene obtained from the library, and wild-type genes for assay crosses. Can be repeated with a single gene shuffled, and with multiple genes each encoding an enzyme with a different activity.

In a modification of this procedure, UDP-glucose in step G was replaced with other NDs.
Substitute with P-sugar. The MS parameters in step H are adapted to detect putative molecular ions.

Example 2 Generation of Methyltransferase Library and Evolution of Erythromycin 6-O-Methyltransferase for Clarithromycin Production This example demonstrates the live production of recombinant O-methyltransferase (OMTase). The production of rallies, as well as the use of enzymes from that library to synthesize derivatives of clarithromycin (6-O-methylerythromycin) is described.

A family of erythromycin analogs having a 6-methoxy group has been shown to have useful pharmaceutical properties. These compounds are currently prepared by multi-step chemical methylation of erythromycin A and its analogs (FIG. 11).
). An enzyme capable of selectively transferring an activated methyl group to a 6-hydroxyl group allows for one step high yield production in vivo of this type of erythromycin analog or single biotransformation in vitro. This example describes an approach for obtaining such a methyltransferase.

At this point no erythromycin 6-OMTase activity was detected. Therefore, it is necessary to generate OMTases of novel specificity. By sampling 10 ⁴ to 10 ⁵ members of the shuffled library, the opportunity to find new activity begins with the naturally occurring sequence rather than random point mutations in the sequence diversity of that library. In some cases, it will be greatly increased. Such libraries span a relatively large portion of the sequence region and are enriched with functional sequences. Thus, DNA shuffling causes OMTas to specifically methylate substrates similar to the 6-hydroxyl of erythromycin.
Perform using a family of homologous genes that encode e. It is uncertain which members of this family are more influential in producing 6-OMTase activity, thus producing a variety of shuffled libraries. For example,
Shuffling each of its subfamilies alone, as well as shuffling the entire family together. This is accomplished using several shuffling forms designed to result in the recombination of both high and low sequence identity genes.

S-adenosylmethionine (SAM) -dependent methyltransferase (MT
) Is a methyl-ester derivative, methyl-ether derivative, methyl-thioether derivative, methyl-amine derivative of proteins, nucleic acids, sugars, polysaccharides, lipids, lignin, and various low molecular weight compounds (for example, macrolides), and Methyl-
It constitutes a class of enzymes that form amide derivatives. SAMs carry activated methyl groups that are efficiently transferred to nucleophiles with a wide range of chemical reactivity. SAM
Transfer of an activated methyl group to a recipient nucleophile is thermodynamically favored, thereby driving this methyl transfer reaction to essentially complete (FIG. 12).
. SAM-dependent OMTa specific for secondary alcohols on carbomycin, midecamycin, saframycin, rapamycin, rifamycin and FK506
A family of seven genes encoding se is known (Fig. 13). Comparison of these substrate nucleophiles with the 6-hydroxy of erythromycin A suggests that only slight regulation in local specificity is required for the parent OMTase to receive erythromycin as a substrate. Another gene of interest is the gene that encodes ERYG, which encodes the mycarose portion of erythromycin C
-Methylation, leading to the synthesis of erythromycin A. EryG shares 54% identity with rapQ at the DNA level, presumably the tertiary alcohol OMTas.
An additional subfamily of OMTases containing e-activity is provided (Figure 14).

The gene to be shuffled is synthesized by PCR from either genomic DNA or synthetic oligonucleotides. Then, these genes are cloned into an appropriate expression vector. The complete sequence of the gene encoding carbomycin-4-OMtase is not known, but is it possible to clone it?
Alternatively, a partial sequence may be shuffled with the complete sequence of another OMTase.

We make several libraries of SAM-dependent OMTases. These libraries were run against 6-OMTa against erythromycin A and its analogs.
Screen for se activity. The identified clones are pooled and further evolved to improve the enzyme to practical levels of activity.

In general, 10 ⁴ ~ from this family shuffled library.
10 ⁵ clones were screened for deserythromycin 6-OMTase
Identify active clones. Cell cultures are grown in the presence of deserythromycin A, then the supernatants of these cultures are removed and assayed for the presence of 6-O-methyldeserythromycin A oxime. The OMTase gene is isolated from the identified clones, pooled, shuffled, and then screened for increased deserythromycin A 6-OMTase activity.
For additional cycles of shuffling and screening, the enzymatic activity of 6
Continue until an appropriate level is reached for the production of -O-methyldeserythromycin.

To ensure the identification of useful activities, the shuffled library was
One may screen for 6-OMTase activity against erythronolide B, deserythromycin A, erythromycin A and their oxime derivatives. What's deserythromycin A oxime 6-OM in the first library
It is possible that no Tase activity will also be detected, although there may be other clones with 6-OMTase activity. These clones can then be used in additional rounds of shuffling to further alter the 6-OMTase specificity. For example, if activity is detected for erythromycin A, subsequent libraries may be first screened for activity for deserythromycin A and finally screened for activity for deserythromycin oxime. In this manner, only subtle changes in specificity are predicted from each new library.

(Generation of Gene and Library) Midecamycin 3'O-methyl transferase (mdmC), safromycin O-methyl transferase (safC), rapamycin 31-O-methyl transferase (rapI) and FK506 31-O-methyl transferase. The gene encoding the open reading frame for (fkbM) (FIG. 13) was isolated and the appropriate E. E. coli expression vector (pE
Clone in T22B (+). Then these genes (50-80
% Identity range) is shuffled by family shuffling to create a library of genes encoding chimeric O-methyl transferases (OMTases). This library was cloned back into an expression vector and the appropriate E. Expression in E. coli host (BL21 (DE3)). Here, the library can be screened for chimeric enzymes with novel properties, such as novel specificity for target methylation.

Generic Screen for OMTase Activity OMTase activity was measured by using an assay that measures the transfer of radiolabeled methyl groups of ( ³ H) S-adenosylmethionine to the desired donor molecule. , Can be measured with high throughput (see FIG. 15). This assay is based on the transfer of labeled methyl groups from highly charged molecules (SAM) to more hydrophobic molecules (Figure 12). The reaction is extracted with an organic solvent such that unreacted SAM remains in the aqueous phase and the methylated substrate is selectively extracted into the organic phase. The organic phase can then be measured for its radioactivity content. The advantage of this assay is that it is generally applicable to extractable substrates, that this assay has very high throughput, and that it can be used to simultaneously screen against pools of compounds for activity. Is. This process is as follows.

Streptomyces lividans is a particularly suitable host for at least two reasons. First, this is an E. It is transformed with high efficiency by the plasmid DNA isolated from E. coli. Second, it is highly permeable to erythromycin and its analogs, and therefore can assay whole cells rather than lysates. Alternatively, Escherichia coli cell extract or B
A high-throughput format can be used to measure enzyme activity from an acillus subtilis cell extract. Purified enzyme or cell lysate was added to an assay mixture (0.4 mM MgSO ₄ , 0.1 m) in 50 mM phosphate buffer (pH 7.5).
MDTT, 0.1 mM ( ³ H) S-adenosylmethionine, and 1-10 m
M target substrate). After incubation, the reaction is quenched by extraction with ethyl acetate. A sample of the organic phase (50 μl) was removed, mixed with scintillant (150 μL), and 9
Radioactivity is measured using a 6-well scintillation counter. Clones from samples with higher radioactivity than control samples without added enzyme are considered positive and can be further investigated in a more quantitative assay.

Evolution of Clarithromycin Synthase Clarithromycin is 6-O-methylerythromycin. The current process for the preparation of clarithromycin is a seven-step chemical methylation of erythromycin. Enzymes that can carry out this chemical reaction in one step may provide a means to prepare clarithromycin by fermentation or biotransformation (see Figure 16). To generate such enzymes, OMTase libraries are screened for erythromycin 6-O methylase activity.

The shuffled OMTase library is plated on solid medium to separate individual clones. Individual clones were added to LB medium (200 μL
) And ampicillin (100 μg / ml) in a 96-well plate. The plates are grown at 30 ° C. for 10 hours or until the culture reaches an optical density of 0.7. Isopropyl thiogalactoside (IPT
G) is added to 0.1 mM to induce expression of the MTase and the cells are incubated for a further 3 hours. Centrifuge the plate and discard the supernatant. The cell pellet was resuspended in lysis buffer (200 μl) in 50 mM phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM DTT, 2 μg / ml polymyxin B sulfate, and 1 mg / ml T4 lysozyme. It becomes cloudy. The reaction is incubated at 30 ° C for 15 minutes.

Samples (20 μl) from each well are transferred to 96 deep well plates containing clarithromycin synthase assay buffer (280 μl) using a 96 head liquid handling station (eg Multimek ^™ ). This buffer solution is 0.4 mM MgSO ₄ , 0.1 mM DTT, 0.1 mM ( ³ H) S.
50 mM phosphate buffer (pH 7.5) containing adenosylmethionine and 1 mM erythromycin. The reaction is incubated at 30 ° C for 1 hour. Ethyl acetate (300 μL) is added to each well, the plate is shaken vigorously, centrifuged, and a sample of the upper organic phase (50 μL) is removed and added to the plate containing scintillant (150 μL). . The plate is then read using a plate scintillation counter. All samples in the organic phase that have a higher radioactivity than those with the parent gene or those without the MTase gene probably contain an enzyme that transfers a methyl group to erythromycin. Since there are 5 hydroxyl groups on the erythromycin that the methyl group could potentially be transferred to, it is necessary to distinguish whether the methyl group was transferred to 6-hydroxyl.

Secondary Assay for Clarithromycin Synthase The secondary assay for clarithromycin synthase activity is based on chemical modification with phenylboronate and analysis by mass spectrometry. Erythromycin is
It can be O-methylated at five positions (positions 6, 11 or 12 of the macrolide ring, or either the cladinose or desosamine moieties). Phenylboronate is a cis diol (eg, 11,12 of erythromycin).
Diol) specifically binds. Thus, when phenylboronate binds to enzymatically methylated erythromycin, the methyl group cannot be located at the 11 or 12 position. The following assay is performed to determine if the modified erythromycin is clarithromycin.

Enzymatic methylation of erythromycin is performed as described above, except that the SAM used for the modification is not radiolabeled and the cell extract is derived from cells showing a positive radioactivity assay. . After extraction from the reaction mixture, the organic phase is washed with 2
Analyze by dimensional mass spectroscopy (MS / MS). In this two-dimensional spectroscopy, the parent ion is fragmented into submolecular fragments (see Figure 17).

Clarithromycin has a cationic molecular weight of 748.48 and the positive charge is due to the protonation of the amine on the desosamine moiety. Upon fragmentation of the clarithromycin cation, cladinose and desosamine can be separated from the macrolide ring, but only the molecule containing the desosamine moiety is detected. Because the molecule is
This is because it possesses an amine. Fragmentation of the 748.48 ion yields two different new ions 590.4 and 158.12. This 590 ion is 6-
O-methyldeserythromycin A (clarithromycin lacking the cladinose moiety). The 158.12 ion is dehydrodesosamine and is the result of the removal of the 5-hydroxy group on the macrolide ring. The MS / MS spectrum of the 748.48 peak with 590 and 158 ions is on the macrolide ring (
That is, it differs from the erythromycin derivative methylated at the 6-, 11- or 12-positions. If the sample shows this spectrum, it is analyzed further to determine if the sample is methylated at the 6-position. The organic extract is treated with excess phenylboronate under neutral conditions and analyzed by mass spectroscopy. Positions 11 and 12 are free to form adducts with phenylboronate only when the modification is in the 6-position. Thus, the presence of molecular ion 834.52 (a phenylboronate adduct of clarithromycin) indicates that the sample contains clarithromycin and that the corresponding clone encodes erythromycin 6-O-methyltransferase.

The examples and embodiments described herein are for illustration purposes only, and various modifications or alterations in light thereof are suggested to those skilled in the art and are intended to be within the spirit and scope of the present application. It is understood that the scope is included within the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference for all purposes.

[0190]   (Sequence list)   (SEQ ID NO: 19: Sequence of pCKZEBB vector)

[0191]

[Table 10]

[Brief description of drawings]

[Figure 1]   FIG. 1 shows potential sugar attachment points on vancomycin hydrochloride.

[Fig. 2]   Figure 2 shows potential sugar attachment points on somatostatin.

[Figure 3]   Figure 3 shows potential sugar attachment points on cholic acid.

[Figure 4]   FIG. 4 shows potential sugar attachment points on L-thyroxine.

[Figure 5]   Figure 5 shows potential sugar attachment points on nogaramycin.

FIG. 6 shows potential sugar attachment points on syringaldazine.

FIG. 7 shows potential sugar attachment points on alcarubicin.

[Figure 8]   FIG. 8 shows potential sugar attachment points on ritodrine hydrochloride.

[Figure 9]   Figure 9 shows potential sugar attachment points on rifamycin.

FIG. 10 shows potential sugar attachment points on ristomycin sulphate. Five additional hydroxyls on this backbone also
Shown (but not shown by arrows); these constitute potential sugar attachment points.

FIG. 11 shows multi-step chemical methylation of erythromycin A and its analogs.

FIG. 12 shows a reaction catalyzed by S-adenosylmethionine (SAM) -dependent methyltransferase.

FIG. 13 shows 6-OM using erythromycin and its analogs as substrates.
Figure 3 shows the specificity of O-methyl transferases that can be shuffled to obtain a recombinant enzyme with Tase activity.

FIG. 14 shows 6-OM using erythromycin and its analogs as substrates.
Figure 3 shows the DNA and protein sequence similarity of O-methyl transferase shuffled to obtain a recombinant enzyme with Tase activity.

FIG. 15 shows the identification of methyltransferases with novel specificity,
Microtiter plate high throughput primary screening.

FIG. 16: Erythromycin A for biocatalytic synthesis of clarithromycin
Figure 3 shows a schematic of the use of 6-O-methyl transferase.

FIG. 17 shows a secondary assay for clarithromycin synthase. 59
MS / MS detection of the 0/158 pair identifies methylation of the macrolide ring.

FIG. 18 shows a further secondary assay for clarithromycin synthase. Phenylboronate reacts specifically with cis diols at neutral pH. Only clarithromycin can react to give the 834.5 ion 11
-12-cis diol.

FIG. 19   FIG. 19 shows a map of the vector pCKZEBB.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Del Cadea, Stephen             United States California 94002,               Belmont, Monroe Avenue             2049 (72) Inventor Serifonov, Sergey A ..             United States California 94024,               Los Altos, Homestead Co             2240 (72) Inventor Howard, Russell             United States California 94022,               Los Altos Hills, Vizcai             Noroad 12700 F-term (reference) 4B024 AA01 AA03 AA20 BA07 BA67                       CA03 CA07 CA20 DA06 EA04                       GA11 GA27 HA01 HA03 HA20                 4B050 CC04 CC05 EE01 FF14E                       GG01 LL01 LL05                 4B064 AF54 CA22 DA01 DA16

Claims (50)

[Claims] 1. A method for obtaining a library of organic molecule derivatives, comprising:
The method comprises contacting an organic molecule with one or more members of a library of recombinant derivatizing enzymes and other necessary reactants to form a library of organic molecule derivatives, Wherein the derivatizing enzyme is: a) modification of one or more functional groups present on the organic molecule; b) modification of a chemical moiety onto one or more functional groups present on the organic molecule. Addition; and c) introducing a new functional group, which catalyzes a reaction selected from the group consisting of: 2. The method of claim 1, wherein the library of organic molecule derivatives comprises one or more members of a second library of recombinant derivatizing enzymes and other requirements. Further reacting the various reactants to form a further library of organic molecule derivatives, wherein the derivatizing enzyme of the second library comprises: a) one of the functional groups One or more modifications; b) catalyzing a reaction selected from the group consisting of the addition of a chemical moiety to one or more of said functional groups; and c) the introduction of new functional groups. 3. The method according to claim 2, wherein the derivatizing enzyme of the second library is a functional group modified or added by the derivatizing enzyme of the first library. Alternatively, a method of catalyzing the addition of a chemical moiety onto the functional group. 4. The method of claim 1, wherein the one or more members of the library of organic molecule derivatives comprises a chemical reaction after the step of contacting the library of recombinant derivatizing enzymes. Alternatively, the method is further derivatized by an enzymatic reaction. 5. The method according to claim 1, wherein the library of recombinant derivatizing enzymes is obtained by the shuffling method. 6. The method of claim 5, wherein the shuffling method comprises:
The following: (1) a step of recombining at least a first form and a second form of a nucleic acid encoding a derivatizing enzyme so as to generate a library of recombinant polynucleotides, wherein The second form differs from each other by two or more nucleotides,
And (2) expressing the library of recombinant polynucleotides so as to obtain the library of recombinant derivatizing enzymes. 7. The method of claim 6, wherein the recombination step is performed in vitro. 8. The method of claim 6, wherein the method further comprises: (3) derivatizing at least one recombinant polynucleotide encoding a member of the library of recombinant derivatizing enzymes. A further form of said nucleic acid encoding a phosphatase and a step of recombining to produce a further library of recombinant nucleic acids, said further form being the same as said first and second forms. Or different; (4) expressing a further library of said recombinant polynucleotides to obtain a further library of recombinant derivatizing enzymes; and (5) said recombination, if necessary. Repeating (3) and (4) until a further library of derivatizing enzymes contains the desired number of different recombinant derivatizing enzymes. , Method. 9. The method of claim 8, wherein at least one recombination step is performed in vitro. 10. The method of claim 5, wherein the shuffling method comprises: (1) performing a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides, one segment of another segment; Starting to produce a population of recombinant polynucleotides under conditions that act as a template for extension; and (2) selecting or screening the recombinant polynucleotides for desired characteristics, Method. 11. The method of claim 10, wherein the overlapping segment is produced by cleavage of the population of variant polynucleotides. 12. The method of claim 11, wherein the cleaving is by DNase I digestion. 13. The method of claim 10, wherein the overlapping segments are produced by chemical synthesis. 14. The method of claim 10, wherein the overlapping segments are produced by amplification of the population of polynucleotides. 15. The method of claim 10, wherein the population of variant polynucleotides is an allelic variant. 16. The method of claim 10, wherein the population of variant polynucleotides is a species variant. 17. The method according to claim 5, wherein the shuffling method is the following: (1) hybridizing at least two nucleic acid sets,
Wherein the nucleic acid set comprises a single-stranded nucleic acid template, and the second nucleic acid set comprises at least one nucleic acid fragment set; and (2) a sequence gap between the hybridized nucleic acid fragments Extending, ligating, or both, so as to produce an at least substantially full-length chimeric nucleic acid sequence corresponding to the nucleic acid template, thereby recombination of the set of nucleic acid fragments. Method. 18. The method according to claim 17, further comprising: (3) denaturing the at least substantially full-length chimeric nucleic acid sequence and the single-stranded nucleic acid template; (4) the at least substantial. Separating the full-length chimeric nucleic acid sequence from the single-stranded nucleic acid template by at least one separation technique; and separating the separated at least substantially full-length chimeric nucleic acid sequence by nuclease digestion or physical fragmentation. Fragmenting to provide a chimeric nucleic acid fragment according to. 19. The method of claim 1, wherein the organic molecule is a lead compound. 20. The method of claim 1, wherein the organic molecule is a naturally occurring compound. 21. The method of claim 1, wherein the organic molecule is a non-naturally occurring compound. 22. The method of claim 1, wherein the members of the library of recombinant derivatizing enzymes are individually contacted with the organic molecule. 23. The method of claim 1, wherein the members of the library of recombinant derivatizing enzymes are subdivided into pools prior to contacting the organic molecule. Method. 24. The method of claim 1, wherein the member of the library of recombinant derivatizing enzymes is contacted with the organic molecule as a mixture of recombinant derivatizing enzymes. 25. The method of claim 1, wherein the recombinant polynucleotide is encoded by a recombinant derivatization of the recombinant polynucleotide by introducing the recombinant polynucleotide into a replicable gene packaging vector. As the enzyme is produced as a fusion with a protein displayed on the surface of a replicable genetic package,
How to express. 26. The method of claim 25, wherein the replicable genetic package is selected from the group consisting of bacteriophage, cells, spores, and viruses. 27. The method according to claim 1, wherein the derivatizing enzyme catalyzes the modification of one or more functional groups on the organic molecule or the functional group is modified by another functional group. A method of catalyzing one or more substitutions therein. 28. The method of claim 27, wherein the functional group is hydrogen and the substitution is with a hydroxyl group. 29. The method of claim 28, wherein the derivatizing enzyme is
A method selected from the group consisting of monooxygenase and dioxygenase. 30. The method of claim 27, wherein the derivatizing enzyme is
A method for catalyzing the introduction of new functional groups onto organic molecules. 31. The method of claim 30, wherein the derivatizing enzyme is
A method selected from the group consisting of halogenases and sulfotransferases. 32. The method of claim 1, wherein the derivatizing enzyme catalyzes the addition of chemical moieties to one or more of the functional groups. 33. The method of claim 32, wherein the derivatizing enzyme is
A method selected from the group consisting of glycosyltransferases, acyltransferases, amidases, methyltransferases, and phosphotransferases. 34. The method of claim 33, wherein the derivatizing enzyme is an acyltransferase and the chemical moiety is a vinyl ester, trihaloethyl, ester, vinyl carbonate, vinyl carbamate, oxime ester. A oxime carbonate, and a bifunctional moiety. 35. The method of claim 33, wherein the derivatizing enzyme is a glycosyltransferase and the chemical moiety is selected from the group consisting of glycosides, aminoglycosides, and glycosid acids. 36. The method of claim 33, wherein the derivatizing enzyme is a glycosyltransferase and the organic molecule is aglycosyl vancomycin hydrochloride, somatostatin, cholic acid, L-thyroxine, nogaramycin, syringalda. A method selected from the group consisting of gin, aclarubicin, ritodrine hydrochloride, rifamycin, and ristomycin sulfate. 37. The method of claim 33, wherein the derivatizing enzyme is O.
A method, which is a methyltransferase and the organic molecule is erythromycin. 38. The method of claim 33, wherein the derivatizing enzyme is amidase and the chemical moiety is selected from the group consisting of amides and peptides. 39. The method of claim 1, further comprising the step of screening a library of organic molecule derivatives to identify those organic molecule derivatives that exhibit the desired properties. 40. The method of claim 39, wherein the desired property is binding to a target molecule. 41. The method of claim 40, wherein the target molecule is selected from the group consisting of receptors, signaling proteins, and ligands.
Method. 42. The method of claim 39, wherein the recombinant derivatizing enzyme is used to identify a member that catalyzes the modification of the organic molecule that imparts the desired property to the resulting organic molecule derivative. The method further comprising screening the members of the library. 43. A method of obtaining an enzyme that catalyzes the synthesis of a desired organic molecule derivative, the method comprising: the step of: adding an organic molecule to a member of a library of recombinant derivatizing enzymes and other necessary reactants. A step of contacting the organic molecule derivative to form a library of organic molecule derivatives; identifying the desired organic molecule derivative in the library of organic molecule derivatives; and the set that catalyzes the synthesis of the desired organic molecule derivative. Identifying a member of the library of recombinant derivatizing enzymes. 44. The method of claim 43, wherein the members of the library of recombinant derivatizing enzymes are individually contacted with the organic molecule. 45. The method of claim 43, wherein the members of the library of recombinant derivatizing enzymes are subdivided into pools prior to contacting the organic molecule. Method. 46. A library of recombinant derivatizing enzymes, wherein when said recombinant derivatizing enzyme is contacted with an organic molecule having one or more functional groups: a) said Catalyze a reaction selected from the group consisting of: modification of one or more of the functional groups; b) addition of a chemical moiety to one or more of the functional groups; and c) introduction of a new functional group. ,Library. 47. The library of claim 46, wherein each of the recombinant derivatizing enzymes comprises multiple blocks of amino acids, the blocks being contiguous in the naturally occurring derivatizing enzyme. Not a library. 48. The library of claim 47, wherein each of said recombinant derivatizing enzymes comprises a block of amino acids resulting from two or more homologues of said derivatizing enzyme. 49. A library of organic molecule derivatives, wherein the library comprises organic molecules having one or more functional groups comprising: a) a modification of one or more of said functional groups; b). Addition of a chemical moiety to one or more of said functional groups; and c) contact with multiple members of a library of recombinant derivatizing enzymes that catalyze a reaction selected from the group consisting of the introduction of new functional groups. A library that is biocatalyst-synthesized by the step of allowing. 50. The library of claim 49, wherein the recombinant derivatizing enzyme comprises: at least a first form and a second form of a nucleic acid encoding the derivatizing enzyme,
Recombining to produce a library of recombinant polynucleotides, wherein the first and second forms differ from each other in two or more nucleotides;
And a step of expressing the library of recombinant polynucleotides to obtain a library of the recombinant derivatizing enzyme.