Industrial Agricultural Biotech – Patricia C. Brzostowicz, Pierre E. Rouviere, EI Du Pont de Nemours and Co

Abstract for “Oxidation using a Brevibacterium Cyclohexanone Monooxygenase of a cyclohexanone-derived cyclohexanone”

An Brevibacterium HCU has identified two gene clusters that encode enzymes for converting cyclohexanol into adipic acids. Each gene cluster has its own open reading frames (ORF) that can be used to produce intermediates in the adipic acids biosynthetic pathway and related molecules. All ORFs have been sequenced. Biochemical analysis and sequence comparison have been used to identify gene function.

Background for “Oxidation using a Brevibacterium Cyclohexanone Monooxygenase of a cyclohexanone-derived cyclohexanone”

The U.S. produced 1.96 billion pounds of adipic acids in 1997, and an additional 2.0 billion in 1998. Adipic acid demand has increased by 2% annually in the past. This trend will continue through 2002, when 1.5-2% of the expected increase is anticipated. Adipic acid is consistently ranked as one of the fifty most popular domestic chemicals. Nylon-6,6 is made from nearly 90% of domestically produced adipic acids. Adipic acid can also be used to produce lubricants, plasticizers, as well as food acids.

The dominant industrial process to synthesize adipic acids is the initial air oxidation cyclohexane. This produces a mixture of cyclohexanone and cyclohexanol (alcohol), which we call KA. (see U.S. Pat. No. 5,221,800). Commercially, hydrogenation of phenol is used to produce KA. However, this process only accounts for 2% of all adipic acids produced. To make adipic acid, KA is produced by both methods. As by-products, reduced nitrogen oxides such as NO2, NO and N2O can be produced and recycled back to nitric acids at different levels.

“Research also has focused on the synthesis of adipic acids from other feedstocks. Carbonylation of butadiene has received a lot of attention (U.S. Pat. No. 5,166,421). Recent research has shown that dimerizing methylacrylates can be done, which opens up the possibility for adipic acid production from C-3 feedstocks.

These processes are difficult to use due to the dependence on environmentally sensitive feedstocks and their tendency to produce undesirable by-products. Adipic acid could be obtained from non-synthetic biological routes. This would be both more beneficial for industry and the environment.

“There are many microbiological routes that can be used. Both wildtype and mutant organisms can convert renewable feedstocks like glucose to adipic acids [Frost John, Chem. Eng. (Rugby, Engl.) (1996), 611, 32-35; WO 9507996; Steinbuechel, AlexanderCLB Chem. Labor Biotech. (1995), 46(6), 277-8; Draths et al., ACS Symp. Ser. (1994), 577 (Benign By Design), 32-45; U.S. Patent. No. No. Similar results have been obtained for organisms with nitrilase activity, which converts nitriles into carboxylic acids, including adipic [Petree et al. AU 669951; and CA 2103616].

“Additionally, wildtype organisms can be used to convert cyclohexane, cyclohexanol, and other alcohols into adipic acid [JP01023894 A2]; Cho, Takeshi, et al. Bio Ind. (1991), 8(10), 671-8; Horiguchi et al., JP 01023895 A2; JP 01023894 A2; JP 61128890 A; Hasegawa et al., Biosci., Biotechnol., Biochem. (1992), 56(8), 1319-20; Yoshizako et al., J. Ferment. Bioeng. (1989), 67(5), 335-8; Kim et al., Sanop Misaengmul Hakhoechi (1985), 13(1), 71-7; Donoghue et al., Eur. J. Biochem. (1975), 60(1), 1-7].”

“One enzymatic pathway for the conversion of cyclohexanol to adipic acid has been suggested as including the intermediates cyclohexanol, cyclohexanone, 2-hydroxycyclohexanone, ?-caprolactone, 6-hydroxycaproic acid, and adipic acid. There are several enzyme activities that can be found in this pathway, such as cyclohexanol oxidase and NADPH-linked NADPH-linked Cyclohexanone Oxase. An alternate enzymatic pathway has been postulated to comprise cyclohexanol?cyclohexanone?1-oxa-2-oxocycloheptane?6-hydroxyhexanoate?6-oxohexanoate?adipate [Donoghue et al., Eur. J. Biochem. (1975), 60(1), 1-7]. The specific sequences of genes encoding the cyclohexanol-to-adipic acid pathway are unknown. This is except for the monoxygenase which is responsible for the conversion from cyclohexanone into caprolactone. [Chen,et., J. Bacteriol., 170, 781-789 (1988)].”

“The solution is to create a synthesis pathway for adipic acids that not only does away with the need to rely on environmentally sensitive starting materials, but also makes use of cheap, renewable resources efficiently. A synthesis route to adipic acids that does not require significant energy inputs, and minimizes toxic by-products, would be very desirable.

“Applicants solved the problem by identifying, isolating, and cloning two monooxygenase genetics, a hydrolase dehydrogenase dehydrogenase dehydrogenase and a cyclohexanol cyclohexanol gene. All of these genes are implicated in the adipic acids biosynthetic pathway.”

“Another embodiment of the invention provides methods to isolate nucleic acids fragments substantially like those that encode polypeptides, as described in SEQID NO.2, SEQID NO.4, SEQID NO.6, SEQID NO.8, SEQID NO:12 and SEQID NO.14, SEQID NO:18, 18, SEQID NO:20, or 22.

“The invention also provides a method of producing adipic acids. It involves: Contacting a transformed host cell with cyclohexanol under suitable growth conditions, where adipic Acid is produced. The transformed host cell contains the nucleic- acid fragments set forth in SEQID NO:15 and 16.”

“The invention also provides methods for producing intermediates in a pathway for the synthesis of adipic acids from cyclohexanol. This includes transformed organisms that have been transformed with any of the open reading frames encoded SEQID NO:4, SEQ ID No:6, SEEQ ID ID NO,8, SEQID NO:12 and SEQID NO:14, respectively, and SEQID NO:22.”

“Additionally, the invention allows for recombinant cell transformations with any gene encoding polypeptides from the group consisting SEQID NO:4, SEEQID NO:6, and SEQID NO:8, as well as SEQID NO:12, SEQID NO:14, EQID NO:18, EQID NO:22, and EQID NO:24.”

“The invention also provides an isolated Brevibacterium SP HCU that contains the genes necessary for the production intermediates of adipic acids as identified by its 16s DNA profile.”

“The present invention contains new sequences that encode key enzymes for the synthesis adipic acids from cyclohexanol. These genes and their expression products can be used to create recombinant organisms capable of producing adipic acids while growing on cyclohexanol, intermediates in the oxidation pathway or for the identification and creation of new bacteria that are capable of producing adipic Acid. The full length sequence of 14 ORFs from two distinct gene clusters has been obtained. Eleven were identified using comparisons to public databases containing protein and nucleotide sequences. This was done using the BLAST algorithm, which is well-known to those who are skilled in the art. Seven ORFs are found in a single gene cluster, here called?gene group 1?. Alternatively, you can call it?GC-1?. This cluster includes ORFs 1.1-1.7. Gene cluster 2 (GC-2), also contains 7 ORFs, which are 2.1-2.7.

“In this disclosure, many terms and abbreviations will be used. These definitions are provided.

“?Open reading frame?” “?Open reading frame” is abbreviated ORF.

“?Polymerase chain reaction? is abbreviated as PCR

“High performance liquidchromatography?” HPLC is abbreviated.

“?Gas chromatography? abbreviated GC

“?Mass spectrometry? MS is an abbreviation of Mass Spectrometry.

“High performance liquid chromatography combined with mass spectrometry?” is abbreviated as LC/MS.

“The term “cycloalkanone derivative” is used. refers to any molecule containing a complete oxidized or derivatized cycloalkanone substructure, including but not limited to cyclobutanone, cyclopentanone, cyclohexanone, 2-methylcyclo-pentanone, 2-methylcyclohexanone, cyclohex-2-ene-1-one, 2-(cyclohex-1-enyl)cyclohexanone, 1,2-cyclohexanedione, 1,3-cyclohexanedione, and 1,4-cyclohexanedione.”

“?HCU? HCU is an abbreviation of?Halophilic Cyclohexanol Utiliser? it is used to identify the Brevibacterium species. “The instant invention”

“The term?adipic acids biosynthetic pathway” This will refer to the enzyme-mediated conversion of cyclohexanol into adipic acids. It also includes the conversion of:

“(1) Cyclohexanol to Cyclohexanone via Cyclohexanol Dehydrogenase”

“(2) cyclohexanone -?-caprolactone via Cyclohexanone Monooxygenase

“(3)??caprolactone to 6-hydroxy-hexanoic Acid via caprolactone Hydrolase”

“(4) 6-hydroxy Hexanoic Acid to 6-aldehyde Hexanoic Acid via 6-hydroxy Hexanoic Acid Dehydrogenase. (5) 6-aldehyde-hexanoic Acid to Adipic acid via 6-aldehydehexanoic Acid Dehydrogenase.

“?Regulator? “Regulator” as it is used herein refers a protein that alters the transcription of a set genes under its control.”

“?Cyclohexanol dehydrogenase? This enzyme catalyzes the conversion from cyclohexanol into cyclohexanone. This enzyme is encoded on GC-1 by either ORF 1.6, ORF 1.7.

“?Cyclohexanone monooxygenase? This enzyme catalyzes cyclohexanone’s conversion to c-caprolactone. This enzyme is encoded using one of two ORFs (resident on GC-1 or GC-2).

“?Caprolactone hydrolase? This enzyme catalyzes conversion of caprolactone into 6-alcohol-hexanoic acids. This enzyme is encoded in ORF 1.2, and is located on GC-1.

“?6-hydroxy hexanoic acid dehydrogenase? This enzyme catalyzes 6-hydroxy hexanoic acids to 6-aldehyde-hexanoic acids. This enzyme is encoded in ORF 2.2, and is located on GC-2.

“Gene cluster” is a term that refers to genes. This will refer to genes that are organized in one expression unit or within close proximity of the chromosome.

“The term?Gene Cluster 1? “The term?Gene cluster 1? or?GC-1? Refers to the 10.6kb gene cluster that contains ORF’s 1.-1.7, which are useful in generating intermediates for the adipic acids biosynthetic pathway.

“The term?Gene Cluster 2? “The term?Gene cluster 2? or?GC-2?” Refers to the 11.5-kb gene cluster that contains ORF 2.1?2.7. This is useful in the generation of intermediates in the Adipic Acid Biosynthetic Pathway.

“An?isolated fragment of nucleic acids? is what we use herein. A single- or two-stranded polymer of DNA or RNA, which may contain synthetic, non-natural, or altered nucleotide base. A nucleic acid fragment that is isolated and forms a polymer of DNA can be made up of one or more segments from cDNA, genetic DNA, or synthetic DNA.

“Adipic acid synthesizing proteins” is the term. The gene product of any sequence in SEQID NO.1, SEQID NO.3, SEQID NO.5, SEQID NO.7, or SEQID NO.13.

“As used herein.?substantially identical? Refers to nucleic acids fragments in which one or more nucleotide base bases are changed. This results in the substitution of one or several amino acids. However, this does not alter the functional properties of the DNA sequence. ?Substantially similar? Also, nucleic acids fragments that are not affected by changes in any of the nucleotide bases but still allow the fragment to mediate the alteration of gene expression using antisense technology or co-suppression technology. ?Substantially similar? Also, the modification of nucleic acids fragments according to the invention is referred to as deletion or addition of nucleotide bases. These modifications do not significantly affect the functional properties or the resulting transcript. The invention covers more than just the exemplary sequences.

“A ?substantial portion? “A?substantial portion?” of an amino acid/nucleotide combination comprising enough of the polypeptide sequence or the nucleotide sequencing of a gene to putatively identity that polypeptide/gen, either manually by one skilled in this art or using computer-automated sequence compari and identification algorithms such as BLAST (“Basic Local AlignmentSearch Tool; Altschul S. F., (1993). J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). A sequence of 10 or more contiguous nucleotides or amino acids is required to identify a polypeptide sequence or nucleic acid sequence that is homologous to a protein or gene. In order to use sequence-dependent methods for gene identification (e.g. Southern hybridization), and isolation (e.g. in situ hybridization (bacteriophage plaques) involving 20-30 contiguous nucleotides, gene specific probes may be used. Short oligonucleotides with 12-15 bases can be used in PCR to amplify a specific nucleic acid fragment. Accordingly, a ?substantial portion? A nucleotide sequence is a portion of the sequence that can be used to identify or isolate a specific nucleic acid fragment. This specification describes partial or complete nucleotide and amino acid sequences that encode one or more fungal proteins. The sequences described herein are available to the skilled artisan. He or she may use any portion of these sequences for purposes that are well-known to those who are skilled in the art. The instant invention includes the complete Sequence Listing and substantial portions of the Sequences as described above.

“The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing. “Complementary” is used to refer to the relationship between nucleotide base that can hybridize to each other. In the case of DNA, for example, adenosine complements thymine, and cytosine complements guanine. The instant invention also contains isolated nucleic acids fragments that complement the complete sequences, as well as the substantially similar sequences.

“Percent identity” is the relationship between two or three polypeptide sequences. “Identity” is used in the art to mean: Identity can also refer to the degree of sequence similarity between polypeptide and polynucleotide sequences as determined by matches between such sequences. ?Identity? Identity and similarity. Can be easily calculated using known methods, including those described in: Computational Molecular Biology (Lesk A. M., ed.). Oxford University Press, New York (1988); BIOCOMPUTING: Informatics and Genome Projects Smith, D. W. (ed.). Academic Press, New York (1993); Computer Analysis of Sequence Data Part I (Griffin A. M. and Griffin H. G., respectively. Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje (G., ed.). Academic Press (1987); Sequence Analysis Primer (Gribskov M. and Devereux J., eds.). Stockton Press New York (1991). The preferred methods for determining identity are those that give the closest match to the sequences being tested. The publically available computer programs contain methods to identify and compare sequences. The GCG Pileup program, which is part of the GCG program package and used in the instant invention to determine identity and similarity among two sequences, is a preferred computer program method. It uses the Needleman algorithm and their standard default values of gap extension penalty=4 and gap creation penalty=12 (Devereux and al., Nucleic Acids Res. 12:387-395 (1984),), BLASTP and BLASTN (Pearson et al. Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988). The BLAST X program can be accessed publicly from NCBI and other sources (BLAST Handbook, Altschul et. al., Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990)). A preferred method to determine percent identity is the DNASTAR protocol which uses the Jotun Hein algorithm. (Hein and al., Methods Enzymol. 183:626-645 (1990)). The default parameters for the Jotun Hein method of alignments are: gap penalty=11, gap length penalty=3, and pairwise alignments ktuple=6. For example, a polynucleotide must have a nucleotide sequencing that has at least 95% identity. The reference nucleotide sequencing of SEQID NO:1 is to be considered identical. However, the polynucleotide may contain up to five point mutations for each 100 nucleotides in the reference nucleotide series of SEQID NO:1. To put it another way, a polynucleotide may have a nucleotide structure at least 95% like a reference nucleotide. However, the reference sequence can contain up to five point mutations per 100 nucleotides. These mutations may occur at the 5 or 3? These mutations may occur at the 5? oder 3? Terminal positions of the reference nucleotide sequencing or anywhere else between them, interspersed either in one or more contiguous group within the reference sequence or individually among nucleotides. A polypeptide with an amino acid sequence that is at least 95% identical to that of SEQID NO:2 is analogous. However, the sequence may contain up to five amino acids alterations for each 100 amino Acids of the reference amino. To put it another way, a polypeptide may have an amino-acid sequence that is at least 95% similar to a reference sequence. However, the sequence can contain up to five amino acid alterations per 100 amino acids of the reference amino acid. These modifications to the reference sequence can occur at the amino and carboxy terminal positions or anywhere else between them, interspersed among the residues of the sequence or within one or more contiguous group.

“?Codon degeneracy? “?Codon degeneracy?” refers to a divergence in genetic code that allows variation of the nucleotide sequencing without affecting the amino acid sequence of an encoded protein. The instant invention refers to any nucleic acids fragment that encodes the entire or a substantial part of the amino acid sequence encoding bacterial adipic Acid Synthesizing Proteins as described in SEQID NO.2, SEQID NO.4, SEQID NO.6, SEQID NO.8, SEQID NO.10, SEQID NO.12, SEQID NO.14, SEQID NO.18, SEQID NO.22, SEQID NO.24 and SEQID NO. The?codon bias? is something that a skilled craftsman is very familiar with. The behavior of a particular host cell when it uses nucleotide codeons to identify a given amino acids. It is important to design a gene that will be more frequently used in the host cell when synthesizing a gene to improve expression.

“?Synthetic genes? Synthetic genes can be made from oligonucleotide-building blocks, which are chemically synthesized according to established procedures. These building blocks can be ligated and annexed to form gene segments, which are then enzymatically assembled into the complete gene. Chemically synthesized, which refers to a sequence or gene, is when the nucleotides have been assembled in vitro. You can either use well-established methods to chemically synthesize DNA or you can automate chemical synthesis using any of the many commercially available machines. The optimization of nucleotide sequencing can allow for optimal gene expression. This is possible to account for the host cell’s codon bias. A skilled artisan can see the potential for successful gene expression when codon usage is biased in favor of the host. A survey of genes from the host cell can help determine which codons are preferred.

“?Gene? Refers to a nucleic acids fragment that expresses a particular protein. This includes regulatory sequences preceding (5?) Non-coding sequences) or following (3? non-coding sequences). The coding sequence. ?Native gene? Refers to a gene found in nature and its own regulatory sequences. ?Chimeric gene? A gene that is not a natural gene and contains regulatory and coding sequences not found in nature. A chimeric gene can contain regulatory sequences or coding sequences from different sources. Or, regulatory sequences may be derived from the same source but are organized in a different way than what is found in nature. ?Endogenous gene? Refers to a native gene that is located in an organism’s genome. A ?foreign? A gene is a gene that is not found in the host but which has been introduced to the host organism through gene transfer. You can have native genes or chimeric gene inserted into non-native genes. A ?transgene? A?transgene? is a gene introduced into the genome through a transformation process.

“?Coding sequence? A DNA sequence that codes specifically for an amino acid sequence. ?Suitable regulatory sequences? Refers to sequences of nucleotides located upstream (5? Non-coding sequences, within or downstream (3? Non-coding sequences), within, or downstream (3? These regulatory sequences can include introns, promoters and translation leader sequences.

“?Promoter? A DNA sequence that can control the expression of a functional RNA or coding sequence. A coding sequence can be found in three places. A promoter sequence is a coding sequence. A promoter sequence can be entirely derived from a native gene or it may contain elements that are derived from other natural promoters. Experts in the field know that promoters can be used to control the expression of genes in different tissue types or cell types. They may also act in different stages of development or respond to different environmental conditions. These are often referred to as “constitutive promotors” because they cause a gene’s expression in all cell types. Furthermore, because the boundaries of regulatory sequences are not always clear, DNA fragments with different lengths could have identical promoter activities.

“?RNA transcript? The product that results from the RNA polymerase catalyzed transcription (RNA transcript) of a DNA sequence. The RNA transcript that is a perfect complement copy of the DNA sequence is called the primary transcript. It may also be referred to by the matureRNA if it is derived from posttranscriptional process of the primary transcript. ?Messenger RNA (mRNA)? The RNA without introns that can be converted into protein by the cells. ?cDNA? A double-stranded, complementary DNA that is derived from mRNA. ?Sense? RNA is an acronym for RNA transcript. It includes the mRNA so that it can be translated into proteins by the cell. ?Antisense RNA? Antisense RNA is an RNA transcript that is complementary to all of the target primary transcripts or mRNAs and which blocks expression of target genes (U.S. Patent. No. 5,107,065). Complementarity between an antisense and specific gene transcripts may occur at any point, i.e. at the 5? non-coding sequence, 3? non-coding sequence, 3? ?Functional RNA? “Functional RNA” is antisense, ribozyme, or any other RNA that has an effect on cellular processes but is not yet translated.

“Operably linked” is a term that refers to the association of nucleic acid sequences on a single nucleic acid fragment. “Operably linked” refers to the association nucleic acids sequences on one nucleic Acid fragment in such a way that their function is affected by each other. A promoter can be operably linked to a coding sequencing if it is capable of changing the expression of that sequence (i.e. the promoter controls the transcription of the coding sequence). It is possible to link coding sequences with regulatory sequences that are either antisense or sense-oriented.

“expression” is the term used to describe the transcription and stable accumulation (mRNA) of sense or antisense genes derived from the nucleic acids fragment of the invention. Expression can also refer to the translation of mRNA into polypeptides.

“?Mature? “?Mature?” ?Precursor? Protein refers to the primary product in translation of mRNA. “Pre- and propeptides can be used to provide intracellular localization signals, but they are not necessarily limited to that.”

“?Transformation? “?Transformation” refers to the incorporation of a nucleic acids fragment into the genome of a host. This results in stable genetic inheritance. Transgenic organisms contain the modified nucleic acids fragments. Or?recombinant or?transformed organisms.”

“The terms ‘plasmid? and?vector?” “The terms?plasmid?,?vector? and?cassette are interchangeable. Extra chromosomal elements are often found in cells that do not have a central metabolism. They usually take the form of circular, double-stranded DNA molecules. These elements can be self-replicating sequences, genome-integrating sequences or nucleotide sequencing sequences. They may also be phage- or nucleotide combinations, either linear or circular, made from single- or multiple-stranded DNA. Untranslated sequences into cells. ?Transformation cassette? A specific vector that contains a foreign gene and has elements that allow for the transformation of a host cell. ?Expression cassette? Refers to a vector that contains a foreign gene, and has elements that enable for the enhanced expression of that foreign gene in a foreign host.”

“Standard recombinantDNA and molecular cloning methods used here are well-known in the art. They are described by Sambrook J., Fritsch E. F. and Maniatis T., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (1989). (hereinafter ‘Maniatis’). ; and Silhavy T. J., Bennan M. L., and Enquist L. W. Experiments With Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor N.Y (1984); and Ausubel F. M. et. al., Current Protocols in Molecular Biology published by Greene Publishing Assoc. Wiley-Interscience (1987).

“The nucleic acids fragments of the present invention can be used to isolate cDNAs or genes encoding homologous proteins from the same or another bacterial species. The art is well-acquainted with the methods for isolating homologous genes by using sequence-dependent protocols. Sequence-dependent protocols are used to isolate homologous genes using sequence-dependent protocols.

“For example, genes that encode similar enzymes to those in the instant adipic acids pathway could be isolated directly using all or part of the instant nucleic acids fragments as DNA hybridization probes. This would allow you to screen libraries for any desired bacteria using a method well-known to those who are skilled in the art. Maniatis demonstrates how to design and synthesize specific oligonucleotide probes that are based on the instant nucleic acids sequences. The entire sequence can also be used to create DNA probes directly using methods that are well-known to skilled artisans, such as random primers DNA Labeling, nick Translation, end-labeling techniques or RNA probes with available in vitro transcript systems. You can also design specific primers to amplify a portion or all of the instant sequences. You can label the amplification products directly in amplification reactions. Or label them after amplification. These probes can be used to identify full-length cDNA and genomic fragments, provided they are stringent enough.

“In addition, two segments of the instant ORFs can be used in polymerase chains reaction protocols to amplify longer nucleic acids fragments encoding homologous gene from DNA orRNA. A library of cloned fragments of nucleic acids may be used to perform the polymerase chain reaction. The sequence of one primer can be derived from instant nucleic acids fragments and the sequences of the two primers take advantage of the existence of the polyadenylic Acid tracts to the 3. End of the mRNA precursor that encodes bacterial genes. The second primer sequence could be derived from sequences obtained from the cloning gene. To generate cDNAs, a skilled artisan could use the RACE protocol (Frohman and al., PNAS USA85:8998 (1988),) to use PCR to amplify copies of the region that lies between the single point in the transcript, and the three? To generate cDNAs, a skilled artisan can use PCR to amplify copies of the region between a single point in the transcript and the 3? oder 5? end. Primers oriented in 3? Primers oriented in the 3? und 5? These sequences can be used to create directions. Commercially available 3? RACE or 5? RACE systems (BRL), 3? Or 5? or 5?

The availability of instant nucleotide sequences and deduced amino acids sequences is a great help in immunological screening cDNA expression library. It is possible to synthesize synthetic peptides that represent portions of the instant amino acids sequences. These peptides may be used to immunize animals and produce monoclonal or polyclonal antibodies that are specific for the amino acid sequences. These antibodies can then be used to screen cDNA Expression Libraries to isolate full-length CDNA Clones of Interest (Lerner R.A. Adv. Immunol. 36:1 (1984); Maniatis).”

“The enzymes and products of the instant ORF may be produced in heterologous hosts cells, especially in cells of microbial host cells. These cells can be used to produce antibodies to the resulting protein using methods that are well-known to those who are skilled in the art. These antibodies can be used to detect the proteins in situ in cells and in vitro in cell extracts. Microbial hosts are the preferred heterologous host cells to produce instant enzymes. Experts in the field are familiar with microbial expression systems and expression vectors that contain regulatory sequences to direct high-level expression of foreign proteins. These could all be used to create chimeric genes that can produce any of the gene products from the instant ORF’s. These chimeric gene could be used to transform microorganisms into high-level expression of the enzymes.

“Chimeric genes can also alter the properties of host bacteria. For example, it is possible to introduce chimeric genes that encode one or more ORF’s 1.2-1.3,1.4,1.4,1.6,1.7,2.2, and 2.4, under the control of appropriate promoters, into host cells comprising at least one of these genes. This will allow the production of various intermediates in adipic acid biosynthetic pathways. The ORF 1.2, which is appropriately controlled, should be expected to produce an enzyme capable to convert?-caprolactone into 6-hydroxy hexanoic acids (FIG). 1). Similar to ORF 2.2 and ORF1.4, it is expected that ORF.2.2 or ORF.1.4 will express an enzyme capable to convert 6-hydroxy hexanoic acids to 6-aldehyde-hexanoic acids (FIG. 1). ORF 1.6 and ORF1.7 could also express an enzyme that converts cyclohexanol into cyclohexanone (FIG. 1). Final, expression of both GC-1 and GC-2 in a single recombinant host will likely result in the conversion of cyclohexanol into adipic acids (FIG. 2).”

ORF 1.3 and ORF 2.4 code the Brevibacterium HCU monooxygenase. Applicant has demonstrated that this monooxygenase, although useful for the conversion of cyclohexanone to ?-caprolactone, has substrate specificity for a variety of other single ring compounds, including, but not limited to cyclobutanone, cyclopentanone, 2-methylcyclopentanone, 2-methylcyclo-hexanone, cyclohex-2-ene-1-one, 2-(cyclohex-1-enyl)cyclohexanone, 1,2-cyclohexanedione, 1,3-cyclohexanedione, and 1,4-cyclohexanedione (see Table 2). The instant monooxygenases could be used to convert any molecule with a complete oxidized/derivatized cyclohexanone structure, such as progesterone and 2-amino-hydroxycaproate.

“It is also possible that open reading frames with high homology to bacterial regulatory element may be helpful in the construction of various expression vectors. ORF’s 1.1, 2.3 and 2.4 each seem to encode a transcriptional regulator. These ORF’s could be used to regulate expression vectors for HiGC Gram-positive bacteria (a group that includes, but is not limited to, the genera Brevibacterium and Corynebacterium as well as Streptomyces and Streptomyces). These vectors could include the gene encoding transcription regulator (repressor, activator), as well as the promoter derived form the upstream sequence of GC-1 and GC-2. The addition of a molecule that induces the cluster would induce transcription. Cyclohexanol, cyclohexanone, or products of their oxidation are likely to be inducers of GC-1 and GC-2 expression.

“Vectors and cassettes that can be used to transform suitable host cells are well-known in the art. The cassette or vector usually contains sequences that direct transcription and translation of relevant genes, a selectable marker and sequences that allow autonomous replication or chromosomal integrtion. Suitable vectors comprise a region 5? A region 5? of the gene that contains transcriptional initiation control controls, and a 3? The DNA fragment that controls transcriptional termination. It is preferable that both control regions be derived directly from the host cells of the same gene. However, it should be noted that these control regions can also be derived from other genes than those of the host species.

The many “Initiation Control Regions or Promoters” that are used to induce expression of the instant ORFs in the desired host cells are well-known to those who are skilled in the art. The present invention is compatible with virtually any promoter that can drive these genes, including, but not limited, CYC1, GAL1, GAL10 and ADH1. PGK and PHO5, GAPDH1, ADC1, TRP1, URA3, LEU2, ENO and TPI (useful expression in Saccharomyces); AOX1 and lac, trp and 1PR, 1PR T7 and trc.

“Termination control areas may also be derived using genes from the preferred host. Although it may not be necessary, it is highly recommended that a termination site be included.

“Description of the Prefer Embodiments”

“The invention concerns the isolation of enzymes that are useful in the conversion of cyclohexanol into adipic acids and the production of enzyme intermediates in the biosynthetic pathway to adipic Acid. Brevibacterium species was used to isolate the relevant genes. It was grown from industrial waste streams. Further study was done on the colonies that were able to grow in halophilic minimal media with cyclohexanone. The 16s rDNA test was used to identify Brevibacterium species HCU. Two gene clusters (GC-1 & GC-2) were identified by RT-PCR and cloned. Sequenced were all open reading frames (ORFs) that reside on both gene clusters. FIG. 2 shows the ORF organization and the possible identification of gene function. 2. Two cyclohexanone monooxygenases encoding ORFs were cloned into expression host genes. Gel electrophoresis confirmed that the genes had been expressed. GC-MS analysis confirmed that the Cyclohexanone monoooxygenase protein expression was active in vitro and in the E. coli host.

“Orf’s 2.2 & 1.4 were also isolated and cloned into E.coli expression hosts for expressions research. GC MS analysis showed that 6-hydroxy hexanoic stimulates the reduction in NADH into NAD, which suggests that both transformants were able to convert 6-hydroxy hexanoic acids to the corresponding aldehyde. These data showed that ORFs 2.2 and1.4 encode 6-hydroxy hexanoic acids dehydrogenase activity.

“The modified RT-PCT protocol for identifying GC-1, GC-2, as well as the relevant open viewing frames, is used to identify GC-1, GC-2, and the appropriate open reading frames. It is based upon the concept of mRNA differentiation display (McClelland et.al., U.S. Pat. No. 5,487,985; Liang, et al., Nucleic Acids Res. (1994), 22(25), 5763-4; Liang et al., Nucleic Acids Res. (1993), 21(14), 3269-75; Welsh et al., Nucleic Acids Res. (1992), 20 (19), 4965-70). This method is particularly useful for the immediate isolation of monooxygenase gene genes because it relies upon the inducibility or message of the gene.

The instant method compares mRNAs obtained by arbitrary RTPCR amplification between control cells and induced cells. A small number of primers are used to generate many bands that can then be analyzed using long-range sequencing gels. This is typically for the analysis of bacterial genes. The Applicant modified the approach by using a larger number of primers, which were analyzed on polyacrylamide-urea gels that are only 15 cm long and 1.5mm thick. These gels are not as clear and have a small length, so faint bands can be difficult to identify. Each primer produces a RAPD pattern that contains ten DNA fragments on average. A set of 81 primers should produce approximately 800 distinct bands, theoretically.

The basic protocol includes 6 steps that follow the growth of cells and totalRNA extraction. These steps include: (i) arbitrarily-primed reverse transcription, PCR amplification; (iii), separation and visualization of DNA products; (v) sequencing of the clones; (vi) identification and analysis of inducible metabolic pathways. The commercial enzyme kit Gibco-BRL Superscript One-Step-RT-PCR System? It contains buffers, the reverse transcriptionase, and the Taqpolymerase in one tube. Each dNTP is 0.4 mM, and MgSO4 is 2.4 mM.

“The primers used were a collection of 81 primers with the sequence 5?-CGGAGCAGATCGAVVVV(SEQ ID NO:38) where VVVV represent all the combinations of the three bases A, G and C at the last four positions of the 3?-end. The 5? The 5?ended sequence was designed to be minimally homologous to both orientations 16S rDNA sequences, many organisms with wide phylogenetic positions. This is to reduce non-specific amplification of these abundant and steady RNA species.”

“The 81 primers had been pre-aliquoted on five plates with 96 well PCR. Each primer was placed on each plate in the indicated two positions.

“A1 A1 A2 A2 A3 A3 A4 A4 A5 A5 A6 A6\nA7 A7 A8 A8 A9 A9 A10 A10 A11 A11 A12 A12\nA13 A13 A14 A14 A15 A15 A16 A16 A17 A17 A18 A18\nA19 A19 A20 A20 A21 A21 A22 A22 A23 A23 A24 A24\nA25 A25 A26 A26 A27 A27 A28 A28 A29 A29 A30 A30\nA31 A31 A32 A32 A33 A33 A34 A34 A35 A35 A36 A36\nA37 A37 A38 A38 A39 A39 A40 A40 A41 A41 A42 A42\nA43 A43 A44 A44 A45 A45 A46 A46 A47 A47 A48 A48”

“Typical RTPCT was performed then using standard protocols well-known in the art.”

“Separation of PCR products was done as follows: 5 % out of every 25?l reaction to RT-PCR were analysed on precutsacrylamide gels (Excell Gels Pharmacia Biotech). Side-by-side analysis of PCR products from Induced RNA and control were done. To visualize the PCR fragments, the gels were stained using the Plus One DNA Silver staining kit (Pharmacia Biotech). The gels were then thoroughly rinsed with distilled water for an hour to remove any acetic acid that was used in the final step of the staining process. Comparison of DNA fragments generated using the same primers in induced and control lanes was made. The scalpel was used to remove bands in the induced lanes but not the control lane.

“Each fragment of reamplified was cloned into pCR2.1-Topo, Invitrogen.

“From the clonings of each differentially expressed bands, four to eight clones were submitted for sequencing with the universal forward. Inserts that failed to yield a complete sequence were sequenced on the opposite strand using the reverse universal primer.

The nucleotide sequences were trimmed to remove primer, vector and low-quality sequences. They were then aligned with the Sequencher program (Gene Code Corporation). The assembled contigs were then compared with the protein and nucleic acids sequence databases using the BLAST align program (BLAST Manual by Altschul et al. Natl. Cent. Biotechnol. Inf:, Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990)).”

After all contigs had been assembled, it was possible to plot the number of clones that each band produced. Many contigs were made up of sequences of identical clones resulting from the cloning a single band. These contigs could be false positives. Other cases may indicate genes that were induced, but only one primer was used in the experiment. Contigs are formed from the alignment DNA sequences taken from different primers.

The following examples further define the invention. These Examples are intended to illustrate preferred embodiments of this invention. These Examples and the discussion above will help one skilled in art to determine the essential characteristics and to make modifications to the invention as needed.

“EXAMPLES”

“General Methods”

“Procedures to phosphorylations (ligations) and transforms are well-known in the art. These techniques can be used in the following examples: Sambrook, J. Fritsch, E. F., and Maniatis T., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y (1989). (hereinafter?Maniatis

“Materials, methods and materials that are suitable for maintaining and growing bacterial cultures are well-known in the art. The following techniques can be used in the following examples: Manual of Methods for General Bacteriology, Phillipp Gerhardt (R. G. E. Murray and Ralph N. Costilow), Eugene W. Nester, Willis A. Wood, Noel R. Krieg, and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994). Thomas D. Brock, Biotechnology: A Textbook on Industrial Microbiology, Second edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). Aldrich Chemicals (Milwaukee), DIFCO Laboratories, Detroit, Mich., GIBCO/BRL, and all materials required for the growth and maintenance bacterial cells were purchased from these companies: Aldrich Chemicals (Milwaukee), DIFCO Laboratories, and DIFCO Laboratories, both in Wisconsin. ), or Sigma Chemical Company, St. Louis, Mo. Except where otherwise noted

“The meanings of abbreviations are as follows:??h? Means hour(s), min? means minute(s), ?sec? means second(s), ?d? means day(s), ?mL? means milliliters, ?L? means liters.”

“Bacterial strains and Plasmids”

“Brevibacterium Sp HCU was isolated by enrichment of activated wastewater from an industrial wastewater treatment plant. E.coli DH5 cells are Max Efficiency capable. GIBCO/BRL (Gaithersburg Md.) purchased DH10B and DH5B. Qiagen (Valencia) purchased expression plasmids pQE30. Cloning vectors pCR2.1, and expression vectors p Trc/His2-Topo have been purchased from Invitrogen (San Diego Calif .).”).

“Growth Conditions:”

“Bacterial cell were grown in Luria Bertani medium, which contains 1% of Bacto-tryptone and 0.5% of Bacto-Yeast Extract. Unless otherwise noted below, 1% of NaCl was used.

“Growth substrates to Brevibacterium species sp. As a sole source of carbon, HCU was added to S12 medium at a concentration of 100ppm.

“Yeast Extract +++\nCasaminoacids +++\nGlucose +\nFructose ++\nMaltose ?\nSucrose ?\nMethanol ?\nEthanol ++\n1-Propanol ++\n2-Propanol ?\n1-Butanol ++\nGlycerol ++\nAcetate +++\nPropionate +++\nButyrate +++\nLactate +++\nSuccinate ++\nDecanoate +\nDecane ?\nHexadecane ?\nPhenol ?\nBenzene ?\nBenzoate ?\nToluene ?\nCyclohexane ?\nCyclohexanone ++\nCyclohexanol +\nCyclopentanone +\nCycloheptanone ?\nCycloheptanol ?\nCyclooctanone ?\nCyclododecanone ?”

“Enzymatic Assays”

“Confirmation that cyclohexanone has been converted to caprolactone was confirmed by GC-Mass Spectrometry using a HP 5890 gas Chromatograph equipped with a HP 5971 mass select detector and a HP-1 capillary columns (Hewlett Packard). Samples were first acidified to pH 3, extracted three times with dichloromethane, dried with MgSO4, and filtered.

Summary for “Oxidation using a Brevibacterium Cyclohexanone Monooxygenase of a cyclohexanone-derived cyclohexanone”

The U.S. produced 1.96 billion pounds of adipic acids in 1997, and an additional 2.0 billion in 1998. Adipic acid demand has increased by 2% annually in the past. This trend will continue through 2002, when 1.5-2% of the expected increase is anticipated. Adipic acid is consistently ranked as one of the fifty most popular domestic chemicals. Nylon-6,6 is made from nearly 90% of domestically produced adipic acids. Adipic acid can also be used to produce lubricants, plasticizers, as well as food acids.

The dominant industrial process to synthesize adipic acids is the initial air oxidation cyclohexane. This produces a mixture of cyclohexanone and cyclohexanol (alcohol), which we call KA. (see U.S. Pat. No. 5,221,800). Commercially, hydrogenation of phenol is used to produce KA. However, this process only accounts for 2% of all adipic acids produced. To make adipic acid, KA is produced by both methods. As by-products, reduced nitrogen oxides such as NO2, NO and N2O can be produced and recycled back to nitric acids at different levels.

“Research also has focused on the synthesis of adipic acids from other feedstocks. Carbonylation of butadiene has received a lot of attention (U.S. Pat. No. 5,166,421). Recent research has shown that dimerizing methylacrylates can be done, which opens up the possibility for adipic acid production from C-3 feedstocks.

These processes are difficult to use due to the dependence on environmentally sensitive feedstocks and their tendency to produce undesirable by-products. Adipic acid could be obtained from non-synthetic biological routes. This would be both more beneficial for industry and the environment.

“There are many microbiological routes that can be used. Both wildtype and mutant organisms can convert renewable feedstocks like glucose to adipic acids [Frost John, Chem. Eng. (Rugby, Engl.) (1996), 611, 32-35; WO 9507996; Steinbuechel, AlexanderCLB Chem. Labor Biotech. (1995), 46(6), 277-8; Draths et al., ACS Symp. Ser. (1994), 577 (Benign By Design), 32-45; U.S. Patent. No. No. Similar results have been obtained for organisms with nitrilase activity, which converts nitriles into carboxylic acids, including adipic [Petree et al. AU 669951; and CA 2103616].

“Additionally, wildtype organisms can be used to convert cyclohexane, cyclohexanol, and other alcohols into adipic acid [JP01023894 A2]; Cho, Takeshi, et al. Bio Ind. (1991), 8(10), 671-8; Horiguchi et al., JP 01023895 A2; JP 01023894 A2; JP 61128890 A; Hasegawa et al., Biosci., Biotechnol., Biochem. (1992), 56(8), 1319-20; Yoshizako et al., J. Ferment. Bioeng. (1989), 67(5), 335-8; Kim et al., Sanop Misaengmul Hakhoechi (1985), 13(1), 71-7; Donoghue et al., Eur. J. Biochem. (1975), 60(1), 1-7].”

“One enzymatic pathway for the conversion of cyclohexanol to adipic acid has been suggested as including the intermediates cyclohexanol, cyclohexanone, 2-hydroxycyclohexanone, ?-caprolactone, 6-hydroxycaproic acid, and adipic acid. There are several enzyme activities that can be found in this pathway, such as cyclohexanol oxidase and NADPH-linked NADPH-linked Cyclohexanone Oxase. An alternate enzymatic pathway has been postulated to comprise cyclohexanol?cyclohexanone?1-oxa-2-oxocycloheptane?6-hydroxyhexanoate?6-oxohexanoate?adipate [Donoghue et al., Eur. J. Biochem. (1975), 60(1), 1-7]. The specific sequences of genes encoding the cyclohexanol-to-adipic acid pathway are unknown. This is except for the monoxygenase which is responsible for the conversion from cyclohexanone into caprolactone. [Chen,et., J. Bacteriol., 170, 781-789 (1988)].”

“The solution is to create a synthesis pathway for adipic acids that not only does away with the need to rely on environmentally sensitive starting materials, but also makes use of cheap, renewable resources efficiently. A synthesis route to adipic acids that does not require significant energy inputs, and minimizes toxic by-products, would be very desirable.

“Applicants solved the problem by identifying, isolating, and cloning two monooxygenase genetics, a hydrolase dehydrogenase dehydrogenase dehydrogenase and a cyclohexanol cyclohexanol gene. All of these genes are implicated in the adipic acids biosynthetic pathway.”

“Another embodiment of the invention provides methods to isolate nucleic acids fragments substantially like those that encode polypeptides, as described in SEQID NO.2, SEQID NO.4, SEQID NO.6, SEQID NO.8, SEQID NO:12 and SEQID NO.14, SEQID NO:18, 18, SEQID NO:20, or 22.

“The invention also provides a method of producing adipic acids. It involves: Contacting a transformed host cell with cyclohexanol under suitable growth conditions, where adipic Acid is produced. The transformed host cell contains the nucleic- acid fragments set forth in SEQID NO:15 and 16.”

“The invention also provides methods for producing intermediates in a pathway for the synthesis of adipic acids from cyclohexanol. This includes transformed organisms that have been transformed with any of the open reading frames encoded SEQID NO:4, SEQ ID No:6, SEEQ ID ID NO,8, SEQID NO:12 and SEQID NO:14, respectively, and SEQID NO:22.”

“Additionally, the invention allows for recombinant cell transformations with any gene encoding polypeptides from the group consisting SEQID NO:4, SEEQID NO:6, and SEQID NO:8, as well as SEQID NO:12, SEQID NO:14, EQID NO:18, EQID NO:22, and EQID NO:24.”

“The invention also provides an isolated Brevibacterium SP HCU that contains the genes necessary for the production intermediates of adipic acids as identified by its 16s DNA profile.”

“The present invention contains new sequences that encode key enzymes for the synthesis adipic acids from cyclohexanol. These genes and their expression products can be used to create recombinant organisms capable of producing adipic acids while growing on cyclohexanol, intermediates in the oxidation pathway or for the identification and creation of new bacteria that are capable of producing adipic Acid. The full length sequence of 14 ORFs from two distinct gene clusters has been obtained. Eleven were identified using comparisons to public databases containing protein and nucleotide sequences. This was done using the BLAST algorithm, which is well-known to those who are skilled in the art. Seven ORFs are found in a single gene cluster, here called?gene group 1?. Alternatively, you can call it?GC-1?. This cluster includes ORFs 1.1-1.7. Gene cluster 2 (GC-2), also contains 7 ORFs, which are 2.1-2.7.

“In this disclosure, many terms and abbreviations will be used. These definitions are provided.

“?Open reading frame?” “?Open reading frame” is abbreviated ORF.

“?Polymerase chain reaction? is abbreviated as PCR

“High performance liquidchromatography?” HPLC is abbreviated.

“?Gas chromatography? abbreviated GC

“?Mass spectrometry? MS is an abbreviation of Mass Spectrometry.

“High performance liquid chromatography combined with mass spectrometry?” is abbreviated as LC/MS.

“The term “cycloalkanone derivative” is used. refers to any molecule containing a complete oxidized or derivatized cycloalkanone substructure, including but not limited to cyclobutanone, cyclopentanone, cyclohexanone, 2-methylcyclo-pentanone, 2-methylcyclohexanone, cyclohex-2-ene-1-one, 2-(cyclohex-1-enyl)cyclohexanone, 1,2-cyclohexanedione, 1,3-cyclohexanedione, and 1,4-cyclohexanedione.”

“?HCU? HCU is an abbreviation of?Halophilic Cyclohexanol Utiliser? it is used to identify the Brevibacterium species. “The instant invention”

“The term?adipic acids biosynthetic pathway” This will refer to the enzyme-mediated conversion of cyclohexanol into adipic acids. It also includes the conversion of:

“(1) Cyclohexanol to Cyclohexanone via Cyclohexanol Dehydrogenase”

“(2) cyclohexanone -?-caprolactone via Cyclohexanone Monooxygenase

“(3)??caprolactone to 6-hydroxy-hexanoic Acid via caprolactone Hydrolase”

“(4) 6-hydroxy Hexanoic Acid to 6-aldehyde Hexanoic Acid via 6-hydroxy Hexanoic Acid Dehydrogenase. (5) 6-aldehyde-hexanoic Acid to Adipic acid via 6-aldehydehexanoic Acid Dehydrogenase.

“?Regulator? “Regulator” as it is used herein refers a protein that alters the transcription of a set genes under its control.”

“?Cyclohexanol dehydrogenase? This enzyme catalyzes the conversion from cyclohexanol into cyclohexanone. This enzyme is encoded on GC-1 by either ORF 1.6, ORF 1.7.

“?Cyclohexanone monooxygenase? This enzyme catalyzes cyclohexanone’s conversion to c-caprolactone. This enzyme is encoded using one of two ORFs (resident on GC-1 or GC-2).

“?Caprolactone hydrolase? This enzyme catalyzes conversion of caprolactone into 6-alcohol-hexanoic acids. This enzyme is encoded in ORF 1.2, and is located on GC-1.

“?6-hydroxy hexanoic acid dehydrogenase? This enzyme catalyzes 6-hydroxy hexanoic acids to 6-aldehyde-hexanoic acids. This enzyme is encoded in ORF 2.2, and is located on GC-2.

“Gene cluster” is a term that refers to genes. This will refer to genes that are organized in one expression unit or within close proximity of the chromosome.

“The term?Gene Cluster 1? “The term?Gene cluster 1? or?GC-1? Refers to the 10.6kb gene cluster that contains ORF’s 1.-1.7, which are useful in generating intermediates for the adipic acids biosynthetic pathway.

“The term?Gene Cluster 2? “The term?Gene cluster 2? or?GC-2?” Refers to the 11.5-kb gene cluster that contains ORF 2.1?2.7. This is useful in the generation of intermediates in the Adipic Acid Biosynthetic Pathway.

“An?isolated fragment of nucleic acids? is what we use herein. A single- or two-stranded polymer of DNA or RNA, which may contain synthetic, non-natural, or altered nucleotide base. A nucleic acid fragment that is isolated and forms a polymer of DNA can be made up of one or more segments from cDNA, genetic DNA, or synthetic DNA.

“Adipic acid synthesizing proteins” is the term. The gene product of any sequence in SEQID NO.1, SEQID NO.3, SEQID NO.5, SEQID NO.7, or SEQID NO.13.

“As used herein.?substantially identical? Refers to nucleic acids fragments in which one or more nucleotide base bases are changed. This results in the substitution of one or several amino acids. However, this does not alter the functional properties of the DNA sequence. ?Substantially similar? Also, nucleic acids fragments that are not affected by changes in any of the nucleotide bases but still allow the fragment to mediate the alteration of gene expression using antisense technology or co-suppression technology. ?Substantially similar? Also, the modification of nucleic acids fragments according to the invention is referred to as deletion or addition of nucleotide bases. These modifications do not significantly affect the functional properties or the resulting transcript. The invention covers more than just the exemplary sequences.

“A ?substantial portion? “A?substantial portion?” of an amino acid/nucleotide combination comprising enough of the polypeptide sequence or the nucleotide sequencing of a gene to putatively identity that polypeptide/gen, either manually by one skilled in this art or using computer-automated sequence compari and identification algorithms such as BLAST (“Basic Local AlignmentSearch Tool; Altschul S. F., (1993). J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). A sequence of 10 or more contiguous nucleotides or amino acids is required to identify a polypeptide sequence or nucleic acid sequence that is homologous to a protein or gene. In order to use sequence-dependent methods for gene identification (e.g. Southern hybridization), and isolation (e.g. in situ hybridization (bacteriophage plaques) involving 20-30 contiguous nucleotides, gene specific probes may be used. Short oligonucleotides with 12-15 bases can be used in PCR to amplify a specific nucleic acid fragment. Accordingly, a ?substantial portion? A nucleotide sequence is a portion of the sequence that can be used to identify or isolate a specific nucleic acid fragment. This specification describes partial or complete nucleotide and amino acid sequences that encode one or more fungal proteins. The sequences described herein are available to the skilled artisan. He or she may use any portion of these sequences for purposes that are well-known to those who are skilled in the art. The instant invention includes the complete Sequence Listing and substantial portions of the Sequences as described above.

“The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing. “Complementary” is used to refer to the relationship between nucleotide base that can hybridize to each other. In the case of DNA, for example, adenosine complements thymine, and cytosine complements guanine. The instant invention also contains isolated nucleic acids fragments that complement the complete sequences, as well as the substantially similar sequences.

“Percent identity” is the relationship between two or three polypeptide sequences. “Identity” is used in the art to mean: Identity can also refer to the degree of sequence similarity between polypeptide and polynucleotide sequences as determined by matches between such sequences. ?Identity? Identity and similarity. Can be easily calculated using known methods, including those described in: Computational Molecular Biology (Lesk A. M., ed.). Oxford University Press, New York (1988); BIOCOMPUTING: Informatics and Genome Projects Smith, D. W. (ed.). Academic Press, New York (1993); Computer Analysis of Sequence Data Part I (Griffin A. M. and Griffin H. G., respectively. Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje (G., ed.). Academic Press (1987); Sequence Analysis Primer (Gribskov M. and Devereux J., eds.). Stockton Press New York (1991). The preferred methods for determining identity are those that give the closest match to the sequences being tested. The publically available computer programs contain methods to identify and compare sequences. The GCG Pileup program, which is part of the GCG program package and used in the instant invention to determine identity and similarity among two sequences, is a preferred computer program method. It uses the Needleman algorithm and their standard default values of gap extension penalty=4 and gap creation penalty=12 (Devereux and al., Nucleic Acids Res. 12:387-395 (1984),), BLASTP and BLASTN (Pearson et al. Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988). The BLAST X program can be accessed publicly from NCBI and other sources (BLAST Handbook, Altschul et. al., Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990)). A preferred method to determine percent identity is the DNASTAR protocol which uses the Jotun Hein algorithm. (Hein and al., Methods Enzymol. 183:626-645 (1990)). The default parameters for the Jotun Hein method of alignments are: gap penalty=11, gap length penalty=3, and pairwise alignments ktuple=6. For example, a polynucleotide must have a nucleotide sequencing that has at least 95% identity. The reference nucleotide sequencing of SEQID NO:1 is to be considered identical. However, the polynucleotide may contain up to five point mutations for each 100 nucleotides in the reference nucleotide series of SEQID NO:1. To put it another way, a polynucleotide may have a nucleotide structure at least 95% like a reference nucleotide. However, the reference sequence can contain up to five point mutations per 100 nucleotides. These mutations may occur at the 5 or 3? These mutations may occur at the 5? oder 3? Terminal positions of the reference nucleotide sequencing or anywhere else between them, interspersed either in one or more contiguous group within the reference sequence or individually among nucleotides. A polypeptide with an amino acid sequence that is at least 95% identical to that of SEQID NO:2 is analogous. However, the sequence may contain up to five amino acids alterations for each 100 amino Acids of the reference amino. To put it another way, a polypeptide may have an amino-acid sequence that is at least 95% similar to a reference sequence. However, the sequence can contain up to five amino acid alterations per 100 amino acids of the reference amino acid. These modifications to the reference sequence can occur at the amino and carboxy terminal positions or anywhere else between them, interspersed among the residues of the sequence or within one or more contiguous group.

“?Codon degeneracy? “?Codon degeneracy?” refers to a divergence in genetic code that allows variation of the nucleotide sequencing without affecting the amino acid sequence of an encoded protein. The instant invention refers to any nucleic acids fragment that encodes the entire or a substantial part of the amino acid sequence encoding bacterial adipic Acid Synthesizing Proteins as described in SEQID NO.2, SEQID NO.4, SEQID NO.6, SEQID NO.8, SEQID NO.10, SEQID NO.12, SEQID NO.14, SEQID NO.18, SEQID NO.22, SEQID NO.24 and SEQID NO. The?codon bias? is something that a skilled craftsman is very familiar with. The behavior of a particular host cell when it uses nucleotide codeons to identify a given amino acids. It is important to design a gene that will be more frequently used in the host cell when synthesizing a gene to improve expression.

“?Synthetic genes? Synthetic genes can be made from oligonucleotide-building blocks, which are chemically synthesized according to established procedures. These building blocks can be ligated and annexed to form gene segments, which are then enzymatically assembled into the complete gene. Chemically synthesized, which refers to a sequence or gene, is when the nucleotides have been assembled in vitro. You can either use well-established methods to chemically synthesize DNA or you can automate chemical synthesis using any of the many commercially available machines. The optimization of nucleotide sequencing can allow for optimal gene expression. This is possible to account for the host cell’s codon bias. A skilled artisan can see the potential for successful gene expression when codon usage is biased in favor of the host. A survey of genes from the host cell can help determine which codons are preferred.

“?Gene? Refers to a nucleic acids fragment that expresses a particular protein. This includes regulatory sequences preceding (5?) Non-coding sequences) or following (3? non-coding sequences). The coding sequence. ?Native gene? Refers to a gene found in nature and its own regulatory sequences. ?Chimeric gene? A gene that is not a natural gene and contains regulatory and coding sequences not found in nature. A chimeric gene can contain regulatory sequences or coding sequences from different sources. Or, regulatory sequences may be derived from the same source but are organized in a different way than what is found in nature. ?Endogenous gene? Refers to a native gene that is located in an organism’s genome. A ?foreign? A gene is a gene that is not found in the host but which has been introduced to the host organism through gene transfer. You can have native genes or chimeric gene inserted into non-native genes. A ?transgene? A?transgene? is a gene introduced into the genome through a transformation process.

“?Coding sequence? A DNA sequence that codes specifically for an amino acid sequence. ?Suitable regulatory sequences? Refers to sequences of nucleotides located upstream (5? Non-coding sequences, within or downstream (3? Non-coding sequences), within, or downstream (3? These regulatory sequences can include introns, promoters and translation leader sequences.

“?Promoter? A DNA sequence that can control the expression of a functional RNA or coding sequence. A coding sequence can be found in three places. A promoter sequence is a coding sequence. A promoter sequence can be entirely derived from a native gene or it may contain elements that are derived from other natural promoters. Experts in the field know that promoters can be used to control the expression of genes in different tissue types or cell types. They may also act in different stages of development or respond to different environmental conditions. These are often referred to as “constitutive promotors” because they cause a gene’s expression in all cell types. Furthermore, because the boundaries of regulatory sequences are not always clear, DNA fragments with different lengths could have identical promoter activities.

“?RNA transcript? The product that results from the RNA polymerase catalyzed transcription (RNA transcript) of a DNA sequence. The RNA transcript that is a perfect complement copy of the DNA sequence is called the primary transcript. It may also be referred to by the matureRNA if it is derived from posttranscriptional process of the primary transcript. ?Messenger RNA (mRNA)? The RNA without introns that can be converted into protein by the cells. ?cDNA? A double-stranded, complementary DNA that is derived from mRNA. ?Sense? RNA is an acronym for RNA transcript. It includes the mRNA so that it can be translated into proteins by the cell. ?Antisense RNA? Antisense RNA is an RNA transcript that is complementary to all of the target primary transcripts or mRNAs and which blocks expression of target genes (U.S. Patent. No. 5,107,065). Complementarity between an antisense and specific gene transcripts may occur at any point, i.e. at the 5? non-coding sequence, 3? non-coding sequence, 3? ?Functional RNA? “Functional RNA” is antisense, ribozyme, or any other RNA that has an effect on cellular processes but is not yet translated.

“Operably linked” is a term that refers to the association of nucleic acid sequences on a single nucleic acid fragment. “Operably linked” refers to the association nucleic acids sequences on one nucleic Acid fragment in such a way that their function is affected by each other. A promoter can be operably linked to a coding sequencing if it is capable of changing the expression of that sequence (i.e. the promoter controls the transcription of the coding sequence). It is possible to link coding sequences with regulatory sequences that are either antisense or sense-oriented.

“expression” is the term used to describe the transcription and stable accumulation (mRNA) of sense or antisense genes derived from the nucleic acids fragment of the invention. Expression can also refer to the translation of mRNA into polypeptides.

“?Mature? “?Mature?” ?Precursor? Protein refers to the primary product in translation of mRNA. “Pre- and propeptides can be used to provide intracellular localization signals, but they are not necessarily limited to that.”

“?Transformation? “?Transformation” refers to the incorporation of a nucleic acids fragment into the genome of a host. This results in stable genetic inheritance. Transgenic organisms contain the modified nucleic acids fragments. Or?recombinant or?transformed organisms.”

“The terms ‘plasmid? and?vector?” “The terms?plasmid?,?vector? and?cassette are interchangeable. Extra chromosomal elements are often found in cells that do not have a central metabolism. They usually take the form of circular, double-stranded DNA molecules. These elements can be self-replicating sequences, genome-integrating sequences or nucleotide sequencing sequences. They may also be phage- or nucleotide combinations, either linear or circular, made from single- or multiple-stranded DNA. Untranslated sequences into cells. ?Transformation cassette? A specific vector that contains a foreign gene and has elements that allow for the transformation of a host cell. ?Expression cassette? Refers to a vector that contains a foreign gene, and has elements that enable for the enhanced expression of that foreign gene in a foreign host.”

“Standard recombinantDNA and molecular cloning methods used here are well-known in the art. They are described by Sambrook J., Fritsch E. F. and Maniatis T., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (1989). (hereinafter ‘Maniatis’). ; and Silhavy T. J., Bennan M. L., and Enquist L. W. Experiments With Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor N.Y (1984); and Ausubel F. M. et. al., Current Protocols in Molecular Biology published by Greene Publishing Assoc. Wiley-Interscience (1987).

“The nucleic acids fragments of the present invention can be used to isolate cDNAs or genes encoding homologous proteins from the same or another bacterial species. The art is well-acquainted with the methods for isolating homologous genes by using sequence-dependent protocols. Sequence-dependent protocols are used to isolate homologous genes using sequence-dependent protocols.

“For example, genes that encode similar enzymes to those in the instant adipic acids pathway could be isolated directly using all or part of the instant nucleic acids fragments as DNA hybridization probes. This would allow you to screen libraries for any desired bacteria using a method well-known to those who are skilled in the art. Maniatis demonstrates how to design and synthesize specific oligonucleotide probes that are based on the instant nucleic acids sequences. The entire sequence can also be used to create DNA probes directly using methods that are well-known to skilled artisans, such as random primers DNA Labeling, nick Translation, end-labeling techniques or RNA probes with available in vitro transcript systems. You can also design specific primers to amplify a portion or all of the instant sequences. You can label the amplification products directly in amplification reactions. Or label them after amplification. These probes can be used to identify full-length cDNA and genomic fragments, provided they are stringent enough.

“In addition, two segments of the instant ORFs can be used in polymerase chains reaction protocols to amplify longer nucleic acids fragments encoding homologous gene from DNA orRNA. A library of cloned fragments of nucleic acids may be used to perform the polymerase chain reaction. The sequence of one primer can be derived from instant nucleic acids fragments and the sequences of the two primers take advantage of the existence of the polyadenylic Acid tracts to the 3. End of the mRNA precursor that encodes bacterial genes. The second primer sequence could be derived from sequences obtained from the cloning gene. To generate cDNAs, a skilled artisan could use the RACE protocol (Frohman and al., PNAS USA85:8998 (1988),) to use PCR to amplify copies of the region that lies between the single point in the transcript, and the three? To generate cDNAs, a skilled artisan can use PCR to amplify copies of the region between a single point in the transcript and the 3? oder 5? end. Primers oriented in 3? Primers oriented in the 3? und 5? These sequences can be used to create directions. Commercially available 3? RACE or 5? RACE systems (BRL), 3? Or 5? or 5?

The availability of instant nucleotide sequences and deduced amino acids sequences is a great help in immunological screening cDNA expression library. It is possible to synthesize synthetic peptides that represent portions of the instant amino acids sequences. These peptides may be used to immunize animals and produce monoclonal or polyclonal antibodies that are specific for the amino acid sequences. These antibodies can then be used to screen cDNA Expression Libraries to isolate full-length CDNA Clones of Interest (Lerner R.A. Adv. Immunol. 36:1 (1984); Maniatis).”

“The enzymes and products of the instant ORF may be produced in heterologous hosts cells, especially in cells of microbial host cells. These cells can be used to produce antibodies to the resulting protein using methods that are well-known to those who are skilled in the art. These antibodies can be used to detect the proteins in situ in cells and in vitro in cell extracts. Microbial hosts are the preferred heterologous host cells to produce instant enzymes. Experts in the field are familiar with microbial expression systems and expression vectors that contain regulatory sequences to direct high-level expression of foreign proteins. These could all be used to create chimeric genes that can produce any of the gene products from the instant ORF’s. These chimeric gene could be used to transform microorganisms into high-level expression of the enzymes.

“Chimeric genes can also alter the properties of host bacteria. For example, it is possible to introduce chimeric genes that encode one or more ORF’s 1.2-1.3,1.4,1.4,1.6,1.7,2.2, and 2.4, under the control of appropriate promoters, into host cells comprising at least one of these genes. This will allow the production of various intermediates in adipic acid biosynthetic pathways. The ORF 1.2, which is appropriately controlled, should be expected to produce an enzyme capable to convert?-caprolactone into 6-hydroxy hexanoic acids (FIG). 1). Similar to ORF 2.2 and ORF1.4, it is expected that ORF.2.2 or ORF.1.4 will express an enzyme capable to convert 6-hydroxy hexanoic acids to 6-aldehyde-hexanoic acids (FIG. 1). ORF 1.6 and ORF1.7 could also express an enzyme that converts cyclohexanol into cyclohexanone (FIG. 1). Final, expression of both GC-1 and GC-2 in a single recombinant host will likely result in the conversion of cyclohexanol into adipic acids (FIG. 2).”

ORF 1.3 and ORF 2.4 code the Brevibacterium HCU monooxygenase. Applicant has demonstrated that this monooxygenase, although useful for the conversion of cyclohexanone to ?-caprolactone, has substrate specificity for a variety of other single ring compounds, including, but not limited to cyclobutanone, cyclopentanone, 2-methylcyclopentanone, 2-methylcyclo-hexanone, cyclohex-2-ene-1-one, 2-(cyclohex-1-enyl)cyclohexanone, 1,2-cyclohexanedione, 1,3-cyclohexanedione, and 1,4-cyclohexanedione (see Table 2). The instant monooxygenases could be used to convert any molecule with a complete oxidized/derivatized cyclohexanone structure, such as progesterone and 2-amino-hydroxycaproate.

“It is also possible that open reading frames with high homology to bacterial regulatory element may be helpful in the construction of various expression vectors. ORF’s 1.1, 2.3 and 2.4 each seem to encode a transcriptional regulator. These ORF’s could be used to regulate expression vectors for HiGC Gram-positive bacteria (a group that includes, but is not limited to, the genera Brevibacterium and Corynebacterium as well as Streptomyces and Streptomyces). These vectors could include the gene encoding transcription regulator (repressor, activator), as well as the promoter derived form the upstream sequence of GC-1 and GC-2. The addition of a molecule that induces the cluster would induce transcription. Cyclohexanol, cyclohexanone, or products of their oxidation are likely to be inducers of GC-1 and GC-2 expression.

“Vectors and cassettes that can be used to transform suitable host cells are well-known in the art. The cassette or vector usually contains sequences that direct transcription and translation of relevant genes, a selectable marker and sequences that allow autonomous replication or chromosomal integrtion. Suitable vectors comprise a region 5? A region 5? of the gene that contains transcriptional initiation control controls, and a 3? The DNA fragment that controls transcriptional termination. It is preferable that both control regions be derived directly from the host cells of the same gene. However, it should be noted that these control regions can also be derived from other genes than those of the host species.

The many “Initiation Control Regions or Promoters” that are used to induce expression of the instant ORFs in the desired host cells are well-known to those who are skilled in the art. The present invention is compatible with virtually any promoter that can drive these genes, including, but not limited, CYC1, GAL1, GAL10 and ADH1. PGK and PHO5, GAPDH1, ADC1, TRP1, URA3, LEU2, ENO and TPI (useful expression in Saccharomyces); AOX1 and lac, trp and 1PR, 1PR T7 and trc.

“Termination control areas may also be derived using genes from the preferred host. Although it may not be necessary, it is highly recommended that a termination site be included.

“Description of the Prefer Embodiments”

“The invention concerns the isolation of enzymes that are useful in the conversion of cyclohexanol into adipic acids and the production of enzyme intermediates in the biosynthetic pathway to adipic Acid. Brevibacterium species was used to isolate the relevant genes. It was grown from industrial waste streams. Further study was done on the colonies that were able to grow in halophilic minimal media with cyclohexanone. The 16s rDNA test was used to identify Brevibacterium species HCU. Two gene clusters (GC-1 & GC-2) were identified by RT-PCR and cloned. Sequenced were all open reading frames (ORFs) that reside on both gene clusters. FIG. 2 shows the ORF organization and the possible identification of gene function. 2. Two cyclohexanone monooxygenases encoding ORFs were cloned into expression host genes. Gel electrophoresis confirmed that the genes had been expressed. GC-MS analysis confirmed that the Cyclohexanone monoooxygenase protein expression was active in vitro and in the E. coli host.

“Orf’s 2.2 & 1.4 were also isolated and cloned into E.coli expression hosts for expressions research. GC MS analysis showed that 6-hydroxy hexanoic stimulates the reduction in NADH into NAD, which suggests that both transformants were able to convert 6-hydroxy hexanoic acids to the corresponding aldehyde. These data showed that ORFs 2.2 and1.4 encode 6-hydroxy hexanoic acids dehydrogenase activity.

“The modified RT-PCT protocol for identifying GC-1, GC-2, as well as the relevant open viewing frames, is used to identify GC-1, GC-2, and the appropriate open reading frames. It is based upon the concept of mRNA differentiation display (McClelland et.al., U.S. Pat. No. 5,487,985; Liang, et al., Nucleic Acids Res. (1994), 22(25), 5763-4; Liang et al., Nucleic Acids Res. (1993), 21(14), 3269-75; Welsh et al., Nucleic Acids Res. (1992), 20 (19), 4965-70). This method is particularly useful for the immediate isolation of monooxygenase gene genes because it relies upon the inducibility or message of the gene.

The instant method compares mRNAs obtained by arbitrary RTPCR amplification between control cells and induced cells. A small number of primers are used to generate many bands that can then be analyzed using long-range sequencing gels. This is typically for the analysis of bacterial genes. The Applicant modified the approach by using a larger number of primers, which were analyzed on polyacrylamide-urea gels that are only 15 cm long and 1.5mm thick. These gels are not as clear and have a small length, so faint bands can be difficult to identify. Each primer produces a RAPD pattern that contains ten DNA fragments on average. A set of 81 primers should produce approximately 800 distinct bands, theoretically.

The basic protocol includes 6 steps that follow the growth of cells and totalRNA extraction. These steps include: (i) arbitrarily-primed reverse transcription, PCR amplification; (iii), separation and visualization of DNA products; (v) sequencing of the clones; (vi) identification and analysis of inducible metabolic pathways. The commercial enzyme kit Gibco-BRL Superscript One-Step-RT-PCR System? It contains buffers, the reverse transcriptionase, and the Taqpolymerase in one tube. Each dNTP is 0.4 mM, and MgSO4 is 2.4 mM.

“The primers used were a collection of 81 primers with the sequence 5?-CGGAGCAGATCGAVVVV(SEQ ID NO:38) where VVVV represent all the combinations of the three bases A, G and C at the last four positions of the 3?-end. The 5? The 5?ended sequence was designed to be minimally homologous to both orientations 16S rDNA sequences, many organisms with wide phylogenetic positions. This is to reduce non-specific amplification of these abundant and steady RNA species.”

“The 81 primers had been pre-aliquoted on five plates with 96 well PCR. Each primer was placed on each plate in the indicated two positions.

“A1 A1 A2 A2 A3 A3 A4 A4 A5 A5 A6 A6\nA7 A7 A8 A8 A9 A9 A10 A10 A11 A11 A12 A12\nA13 A13 A14 A14 A15 A15 A16 A16 A17 A17 A18 A18\nA19 A19 A20 A20 A21 A21 A22 A22 A23 A23 A24 A24\nA25 A25 A26 A26 A27 A27 A28 A28 A29 A29 A30 A30\nA31 A31 A32 A32 A33 A33 A34 A34 A35 A35 A36 A36\nA37 A37 A38 A38 A39 A39 A40 A40 A41 A41 A42 A42\nA43 A43 A44 A44 A45 A45 A46 A46 A47 A47 A48 A48”

“Typical RTPCT was performed then using standard protocols well-known in the art.”

“Separation of PCR products was done as follows: 5 % out of every 25?l reaction to RT-PCR were analysed on precutsacrylamide gels (Excell Gels Pharmacia Biotech). Side-by-side analysis of PCR products from Induced RNA and control were done. To visualize the PCR fragments, the gels were stained using the Plus One DNA Silver staining kit (Pharmacia Biotech). The gels were then thoroughly rinsed with distilled water for an hour to remove any acetic acid that was used in the final step of the staining process. Comparison of DNA fragments generated using the same primers in induced and control lanes was made. The scalpel was used to remove bands in the induced lanes but not the control lane.

“Each fragment of reamplified was cloned into pCR2.1-Topo, Invitrogen.

“From the clonings of each differentially expressed bands, four to eight clones were submitted for sequencing with the universal forward. Inserts that failed to yield a complete sequence were sequenced on the opposite strand using the reverse universal primer.

The nucleotide sequences were trimmed to remove primer, vector and low-quality sequences. They were then aligned with the Sequencher program (Gene Code Corporation). The assembled contigs were then compared with the protein and nucleic acids sequence databases using the BLAST align program (BLAST Manual by Altschul et al. Natl. Cent. Biotechnol. Inf:, Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990)).”

After all contigs had been assembled, it was possible to plot the number of clones that each band produced. Many contigs were made up of sequences of identical clones resulting from the cloning a single band. These contigs could be false positives. Other cases may indicate genes that were induced, but only one primer was used in the experiment. Contigs are formed from the alignment DNA sequences taken from different primers.

The following examples further define the invention. These Examples are intended to illustrate preferred embodiments of this invention. These Examples and the discussion above will help one skilled in art to determine the essential characteristics and to make modifications to the invention as needed.

“EXAMPLES”

“General Methods”

“Procedures to phosphorylations (ligations) and transforms are well-known in the art. These techniques can be used in the following examples: Sambrook, J. Fritsch, E. F., and Maniatis T., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y (1989). (hereinafter?Maniatis

“Materials, methods and materials that are suitable for maintaining and growing bacterial cultures are well-known in the art. The following techniques can be used in the following examples: Manual of Methods for General Bacteriology, Phillipp Gerhardt (R. G. E. Murray and Ralph N. Costilow), Eugene W. Nester, Willis A. Wood, Noel R. Krieg, and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994). Thomas D. Brock, Biotechnology: A Textbook on Industrial Microbiology, Second edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). Aldrich Chemicals (Milwaukee), DIFCO Laboratories, Detroit, Mich., GIBCO/BRL, and all materials required for the growth and maintenance bacterial cells were purchased from these companies: Aldrich Chemicals (Milwaukee), DIFCO Laboratories, and DIFCO Laboratories, both in Wisconsin. ), or Sigma Chemical Company, St. Louis, Mo. Except where otherwise noted

“The meanings of abbreviations are as follows:??h? Means hour(s), min? means minute(s), ?sec? means second(s), ?d? means day(s), ?mL? means milliliters, ?L? means liters.”

“Bacterial strains and Plasmids”

“Brevibacterium Sp HCU was isolated by enrichment of activated wastewater from an industrial wastewater treatment plant. E.coli DH5 cells are Max Efficiency capable. GIBCO/BRL (Gaithersburg Md.) purchased DH10B and DH5B. Qiagen (Valencia) purchased expression plasmids pQE30. Cloning vectors pCR2.1, and expression vectors p Trc/His2-Topo have been purchased from Invitrogen (San Diego Calif .).”).

“Growth Conditions:”

“Bacterial cell were grown in Luria Bertani medium, which contains 1% of Bacto-tryptone and 0.5% of Bacto-Yeast Extract. Unless otherwise noted below, 1% of NaCl was used.

“Growth substrates to Brevibacterium species sp. As a sole source of carbon, HCU was added to S12 medium at a concentration of 100ppm.

“Yeast Extract +++\nCasaminoacids +++\nGlucose +\nFructose ++\nMaltose ?\nSucrose ?\nMethanol ?\nEthanol ++\n1-Propanol ++\n2-Propanol ?\n1-Butanol ++\nGlycerol ++\nAcetate +++\nPropionate +++\nButyrate +++\nLactate +++\nSuccinate ++\nDecanoate +\nDecane ?\nHexadecane ?\nPhenol ?\nBenzene ?\nBenzoate ?\nToluene ?\nCyclohexane ?\nCyclohexanone ++\nCyclohexanol +\nCyclopentanone +\nCycloheptanone ?\nCycloheptanol ?\nCyclooctanone ?\nCyclododecanone ?”

“Enzymatic Assays”

“Confirmation that cyclohexanone has been converted to caprolactone was confirmed by GC-Mass Spectrometry using a HP 5890 gas Chromatograph equipped with a HP 5971 mass select detector and a HP-1 capillary columns (Hewlett Packard). Samples were first acidified to pH 3, extracted three times with dichloromethane, dried with MgSO4, and filtered.

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