WO2007046943A2 - Method of producing biotin - Google Patents

Method of producing biotin Download PDF

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Publication number
WO2007046943A2
WO2007046943A2 PCT/US2006/032733 US2006032733W WO2007046943A2 WO 2007046943 A2 WO2007046943 A2 WO 2007046943A2 US 2006032733 W US2006032733 W US 2006032733W WO 2007046943 A2 WO2007046943 A2 WO 2007046943A2
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biotin
seq
host cell
dna
plasmid
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PCT/US2006/032733
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French (fr)
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WO2007046943A3 (en
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Beatrice A. Clack
Alan B. Youngblood
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Stephen F. Austin State University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
    • C12P17/186—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system containing a 2-oxo-thieno[3,4-d]imidazol nucleus, e.g. Biotin

Definitions

  • the present invention relates to the production of biotin using genetically engineered organisms.
  • Biotin (vitamin B 8 or vitamin H) is a nonpolypeptide coenzyme molecule involved in enzyme-catalyzed reactions requiring carboxyl group transfers. Biotin, like many coenzymes, cannot be synthesized by animals and must instead be obtained exogenously from plants or microorganisms in the diet. Large-scale production of biotin for commercial use as a dietary supplement is therefore desirable. Genetically-modified microorganisms may produce dietary supplements in commercially advantageous amounts and the present invention provides an approach to accomplish this goal for biotin.
  • biotin in microorganisms is achieved by both chemical and fermentation methods.
  • microbial synthesis of biotin in vivo is driven from an operon containing a cluster of genes.
  • the arrangement of genes within the cluster is specific to each bacterial species.
  • the concentration of intermediates and product, as well as the amount of biotinylated protein in a cell regulates biotin operon transcriptional activity (Weaver et al., 2001). For instance, repression of the operon occurs through binding of the birA gene product together with biotinoyl-AMP to the regulatory sequence that lies between the bioA gene and the biotin operon (Weaver et al., 2001; Brown and Kamogawa, 1991).
  • Biotin synthesis requires the proteins encoded by the bioH, bioC, bioF, bioA, bioD, and bioB genes.
  • bioH, bioC, bioF, bioA, bioD, and bioB genes encoded by the proteins encoded by the bioH, bioC, bioF, bioA, bioD, and bioB genes.
  • the steps are predicted to include converting (1) pimelic acid to pimelyl-CoA (PmCoA) by the bioC gene product, which is unidentified, and pimeloyl CoA synthetase that is encoded by bioH (Ploux et al., 1992); (2) Pimelyl-CoA to 7-keto-8-amino pelargonic acid (KAPA) by 7-KAP synthetase (BioF); (3) KAPA to 7,8-diamino-pelargonic acid (DAPA) by DAPA aminotransferase (BioA); (4) DAPA to dethiobiotin by dethiobiotin synthetase (BioD); and (5) dethiobiotin to biotin by biotin synthetase (BioB). See FIG. 1. Synthesis of PmCoA reportedly involves different enzymatic steps in different microorganisms. (Bower et al., 1996)
  • the biotin operon for E.coli consists of a 5.8 Kb region containing five biotin operon genes, bioA, bioB, bioF, bioC and bioD (Otsuka et al., 1988).
  • the bioA gene runs in the opposite direction with control of the operon being between the bioA and bioB sequences, basepairs 807,191 through 812,170 for E.coli K12 (NCBI accession number: NC_000913).
  • the regulatory region is shared by bioA and the rest of the cassette having two promoters running in the opposite direction and on either side of the operator (Brown and Kamogawa, 1991). BioH, in E.
  • the present invention overcomes these deficiencies through the combination of specific genes whose encoded products are involved in biotin synthesis. Furthermore, the present invention discloses the creation of a mutant strain of Pseudomonas mutabilis that produces gram per liter amounts of biotin when transformed with a unique synthetic operon engineered according to the teachings disclosed herein.
  • the present invention relates to biotin biosynthesis in transformed Pseudomonas mutabilis and Escherichia coli. Chromosomal DNA fragments carrying biotin biosynthetic genes responsible for biotin biosynthesis were cloned and engineered to increase biotin production, in part, through the unique use of uniquely applied promoter sequences.
  • the present invention further relates to Pseudomonas strains in which at least one gene involved in biotin biosynthesis is reoriented from its natural 5'-3 ' orientation, and also to the production process of biotin by this genetically engineered P. mutabilis strain.
  • DNA fragments mentioned above may be of various origins, it is preferable to use the strains belonging to the genus Pseudomonas and in particular, P. aeruginosa.
  • FIG. 1 is the biotin biosynthetic pathway predicted in Pseudomonas, including P. mutabilis.
  • FIG. 2 is the plasmid structure for the p519gfp plasmid used to assess PfI phage promoter strength and that served as the foundation plasmid in embodiments of the present invention.
  • FIG. 3 shows the p519gfp plasmid structures used according to embodiments of the present invention with restriction sites identified.
  • FIG. 3 A shows the p519gfp plasmid with the G8 promoter.
  • FIG. 3B shows the p519gfp plasmid with the consensus promoter (Rcon).
  • FIG. 3C shows the p519 plasmid with the IR promoter.
  • FIG. 4 is a partial PfI chromosomal map showing G8 and IR PfI promoters with restriction sites and PCR primer sequences.
  • FIG. 5 shows PCR amplification of the PfI phage G8 (lane 2) and IR (lane 3) promoter PCR products. Lanes 1 and 4 are 100-bp ladders.
  • FIG. 6 is a restriction and gene organization map of pCYIR2-2 (6.4 Kb), also referred to as "IR2- 2 DNA,” of the bio genes according to at least one embodiment of the present invention.
  • FIG. 7 shows the purified 4.9 Kb PCR product (lane 2) from P. aeruginosa containing the bioBFHCD cassette. Lambda HindIII markers are in lane 1.
  • FIG. 8 is the plasmid structure with insertion of the bioBFHCD cassette annealed into the pET- 30 EKJLlC plasmid with restriction sites identified.
  • FIG. 8 A shows the linearized vector/plasmid cleaved at the LIC site.
  • FIG. 8B shows the bioBFHCD cassette inserted into the pET-30 EK/LIC plasmid.
  • FIG. 9 is a comparison of colonies 14 and 15 screened for proper ligation of the bioBFHCD biotin cassette into the pET-30/LIC plasmid. ⁇ Hindlll markers are shown in lanes 1 and 10. Colony 14 DNA is shown in lanes 2 through 5. Colony 14 DNA was undigested (lane 2), or digested with Xbal (lane 3), EcoRl (lane 4), or double-digested with Xbal and EcoRl (lane 5). Colony 15 DNA is shown in lanes 6 through 9. Similarly, Colony 15 DNA was undigested (lane 6), or digested with Xbal (lane 7), EcoRl (lane 8), or double-digested with Xbal and EcoRl (lane 9). The 4.9 Kb bioBFHCD cassette for Colony 15 (lane 9) indicates proper insertion of the cassette into the plasmid.
  • FIG. 10 shows DNA integrity of Colony 15 expression vector/cassette DNA transformed into competent One ShotTM E. coli in, as examples, three of the five selected colonies. All colonies exhibited DNA banding identical to the original DNA of Colony 15. HindIII markers are shown in lanes 1 and 11. DNA was undigested (lanes 2, 5 and 8), digested with Xbal (lanes 3, 6 and 9), or double-digested with Xbal and EcoRl (lanes 4, 7 & 10). All three showed to have the correct insert at 4.9 Kb. All colony DNA exhibited banding identical to the original DNA of Colony 15.
  • FIG. 11 shows plasmid structures with restriction sites and further showing the insertion of the bioA sequence (1.5 Kb) into the pCRII-TOPO plasmid (4 Kb) to form the pCRII-TOPO-BioA plasmid (5.5 Kb).
  • FIG. 1 IA shows the bioA sequence in its normal chromosomal orientation and the final orientation after amplification by PCR and for insertion into the pCRII-TOPO plasmid which is shown in FIG. 1 IB.
  • FIG. 11C shows the final pCRII-TOPO-bioA plasmid.
  • FIG. 12 shows plasmid structures that show the insertion of the bioA sequence (1.5 Kb) in the same orientation and upstream of the bioBFHCD cassette annealed in the pET-30/LIC vector to form the pET-30/LIC-bio ABFHCD plasmid.
  • FIG. 12A shows the pET-30 EK/LIC-bioBFHCD ⁇ lasmid (10.3 Kb).
  • FIG. 12B shows the pCRII-TOPO-bioA plasmid (1.5 Kb).
  • FIG. 12C shows the final pET-30/LIC-bio ABFHCD plasmid (approximately 11.7 Kb).
  • FIG. 13 shows the PCR product of bioA.
  • DNA shown was excised and purified for ligation into the pET-30/LIC-bioBFHCD vector. Shown are ⁇ Hindlll markers (lane 1) and the same bio A PCR product in multiple lanes (lanes 2, 3 and 4).
  • FIG. 14 is a photograph of gel-purified Xbal/Ndel bioA DNA (Lane 2) and 3ofl5 DNA (Lane 3). Lane 1 : ⁇ Hindlll markers.
  • FIG. 15 is a photograph showing correct insertion of gel-purified bio A into the pCRII-TOPO vector in three colonies. ⁇ Hindlll markers are shown in Lane 1. Colony 1 is shown in Lanes 2 and 3. Colony 2 is shown in Lanes 4 and 5. Colony 3 is shown in lanes 6 and 7. DNA was digested with Xbal/Ndel to verify the 1.5 Kb dropout corresponding to the bioA insert.
  • FIG. 16 is a photograph of a DNA gel confirming ligation of the bio ABFHCD cassette into the pET-30/LIC vector. ⁇ Hindlll DNA markers are shown in lanes 1 and 14.
  • Colony 50 DNA was undigested (lane 2) or digested with Xbal (lane 3), Ndel, (lane 4), or double digested with Xbal and Ndel (lane 5).
  • Colony 51 DNA was also undigested (lane 6), digested with Xbal (lane 7) or Ndel (lane 8) or double-digested with Xbal and Ndel (lane 9).
  • Colony 52 DNA was undigested (lane 10), digested with Xbal (lane 11) or Ndel (lane 13) or double-digested with Xbal and Ndel (lane 13).
  • Colony 52 shows the expected banding.
  • FIG. 18 shows the final construct termed pCYIR2-2 with the bioABFHCD cassette driven by the PfI IR promoter and used according to one embodiment of the invention.
  • FIG. 20 is a photograph of a 12% SDS-PAGE gel showing production of recombinant biotin synthesis enzymes coded by the IR2-2 cassette (see FIG. 6) expressed in the 1F9 P. mutabilis mutants.
  • FIG. 21 is a spectrophotograph demonstrating diminished absorbance corresponding to the oxidation of NADPH to NADP that reflects biotin synthase (BioB) activity and biotin production in cell free lysates of mutant 1F9 Pseudomonas mutabilis expressing the bioABFHCD cassette of the IR2-2 plasmid.
  • BioB biotin synthase
  • Induced IPTG-induced cell extract containing reaction mix (without SAMe) was added.
  • “Induced plus” consisted of IPTG induced extract plus SAMe added to the reaction mix.
  • FIG. 23 shows biotin production in excess of six grams per liter assessed by the DACA assay of 1F9 P. mutabilis mutants, transformed with the IR2-2 plasmid that encodes the bioABFHCD cassette, grown in continuous culture.
  • FIG. 24 is a HPLC chromatogram showing the amount of biotin produced by the 1F9 P. mutabilis cells transformed with the IR2-2 plasmid containing the bioABFHCD cassette in an embodiment of the present invention.
  • Panel A is a trace of supernatant.
  • Panel B shows standards of biotin and d-dethiobiotin. Panels A and B are the same scale.
  • the present invention provides a novel genetic construct that comprises a unique arrangement of the complete collection of genes that express proteins necessary for biotin synthesis in Pseudomonas.
  • Bacteriophage PfI promoters that natively drive expression of coat proteins or drives transcription of other viral proteins were cloned and operably linked to the biotin construct to create a unique operon that overproduces biotin in P. mutabilis.
  • Pseudomonas produces biotin and biotin production is self controlled via a negative feedback mechanism.
  • P. mutabilis mutants were created that constitutively produce biotin.
  • Biotin genes bioB, bioF, bioH, bioC, and bioD were cloned from P. aeruginosa as a single primary cassette.
  • BioA is normally found in the reverse direction but 5-prime to the bioBFHCD cluster, but in the practice of the present invention, was reoriented in the same direction but maintained upstream 5-prime to the bioBFHCD cluster, or primary cassette.
  • bioABFHCD cassette was inserted under the selected bacteriophage promoter cloned previously to form a complete cassette and transformed into the P. mutabilis mutant, resulting in the profound overproduction of biotin and highly efficient conversion of dethiobiotin to biotin.
  • An "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • a "nucleic acid molecule construct” is a nucleic acid molecule, either single- or double- stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear or linearized DNA denotes non-circular DNA molecules having free 5' and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • RNA or DNA can be either cDNA or genomic DNA.
  • Polynucleotide probes and primers are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
  • PCR primers are at least 5 nucleotides in length, preferably 15 or more nucleotides, more preferably 20-30. Short polynucleotides can be used when a small region of the gene is targeted for analysis.
  • Promoter refers to a nucleotide sequence comprising a regulatory element that drives gene expression, for example, in an expression vector.
  • Several types of promoters are now well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known.
  • an "expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to” the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a "recombinant host” or “host cell” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell from E. coli or Pseudomonas that produces biotin biosynthetic enzymes from an expression vector.
  • biotin biosynthetic enzymes can be produced by a cell that is a "natural source" of biotin biosynthetic enzymes, and that lacks an expression vector. Production of Pseudomonas mutabilis mutant
  • P. mutabilis was grown in 1.0 L of Difco 0001 media 48 hours in a 37° C shaker incubator at 200 rpm. Eight 50 mL conical tubes with 50 mL of culture were collected and centrifuged at 3000 rpm for 20 minutes to compact cells and 100 mLs of culture was reserved for later use. The centrifuged cells were resuspended in 25 mL of 0.01 M potassium phosphate buffer then centrifuged for an additional 20 minutes at 3000 rpm. The supernatant was discarded and the cells were washed twice with buffer and then resuspended in 1OmL of 0.01 M potassium phosphate buffer containing 0.1 mg/mL of nitrosoguanidine.
  • the cells were gently resuspended and allowed to incubate for 15 minutes in a 37° C water bath without shaking. After incubation the cells were centrifuged as before, the supernatant was removed and stored for disposal. The cells were washed twice as before with 0.01 M potassium phosphate buffer. The pelleted cells were then resuspended in 10 mL of Difco 0001 media containing 20% glycerol and transferred to cryotubes in 1.0 mL aliquots, snap frozen in liquid nitrogen, and stored at -70° C for later use. One tube was stored at -20° C for immediate use.
  • Difco 0001 media 600 mL
  • nitrosoguanidine 0.01 mg/mL of nitrosoguanidine was added to the reserved culture (100 mL). This culture was allowed to grow for 50 hours in a 37° C shaker incubator at 200 rpm. Cells were processed as with acute mutagenesis protocol with nitrosoguanidine except, due to high cell density, 30 mL of cryobuffer was used for final resuspension. Cells were aliquoted at 2 mL per cryotube, snap frozen in liquid nitrogen, and stored at -70° C.
  • EtBr ethidium bromide
  • mutabilis culture was added to 500 mL of Difco 0001 media. The culture was allowed to grow until turbid and EtBr was added to a concentration of 10 ⁇ g/mL. The culture was exposed to UV light at 336 ran for 5 hours and then allowed to grow overnight at 37° C as previous. The cells were harvested and stored.
  • BM-I agar plates were prepared and allowed to dry at 37° C. To each plate 200 ⁇ L (100 ⁇ g/mL) of each of four biotin analogues was applied. The analogue application was allowed to soak into the media for four hours at room temperature protected from light. A 10 ⁇ 2 dilution (100 ⁇ L) of acute mutagenic cells of P. mutabilis (previously prepared) was spread onto each plate for a total often plates for each of the four biotin analogues: biotin methyl ester, biotin p- nitrophenyl, 4-amido-benzoic acid, and diamino biotin. These plates were incubated overnight at 28° C.
  • HABA 4-Hydroxyazobenzene-2-carboxylic acid
  • HABA was obtained from Sigma/Aldrich and the protocol was followed according to the information provided. (Sigma/Aldrich, Product No. H2153 referencing Green NM, 1970). The range in AbS 500 change is between 0.1 -0.4.
  • a scaled down reaction was utilized on a 96 well microtiter plate. In each case 10 ⁇ L of previously prepared and stored supernatant was added to 90 ⁇ L of HABA/Avidin reagent. Phosphate-buffered saline (PBS) IX was used as a diluent. For each of the 96 well plates, triplicate biotin standards were applied.
  • PBS Phosphate-buffered saline
  • the standards were applied in the following gradient: 1.0 ⁇ g/mL, 2.5 ⁇ g/mL, 5.0 ⁇ g/mL, 10 ⁇ g/mL, 25 ⁇ g/mL, and 50 ⁇ g/mL.
  • triplicate samples of HABA only and HABA plus 10 ⁇ L of Ml media only were used as blanks and background, respectively.
  • the p519gfp plasmid served as the foundation plasmid using green fluorescent protein (GFP) to report promoter strength (ATCC 87453; Matthysse et al, 1996).
  • the GFP gene was excised from the final construct.
  • the p519gfp plasmid in host bacteria was obtained from the American Type Culture Collection, Accession No. 87452 (lot 1178894) (P.O. Box 1549, Manassas, VA 20108).
  • the structure of the p519gfp plasmid is shown in FIG. 2. Xbal and EcoRI restriction sites border the GFP gene within the p519gfp plasmid.
  • Xbal and EcoRI were used to replace the GFP gene with the biotin cassette, discussed below.
  • the lyophilized p519gfp plasmid was resuspended in 500 ⁇ L of phosphate-buffered saline (PBS) containing 20% glycerol.
  • PBS phosphate-buffered saline
  • the suspension was aliquoted (50 ⁇ L), snap frozen in liquid nitrogen, and stored at - 70° C for later use.
  • One tube was used to make dilutions and plated onto LB plated containing 50 ⁇ g/mL of kanamycin (Kan). Proper insertion of the promoter sequence conveys kanamycin resistance of the host cell. These plates were placed into a 37° C incubator overnight.
  • Filamentous bacteriophages such as PfI, Pf3, fd, M13, XfI /IfI and Ike produce thousands of copies of G8 coat protein that form a protein capsid surrounding a single copy of single-stranded circular DNA, and each of the DNAs for these viral phages contains an intergenic region (IR) responsible for replication and transcription of other viral proteins.
  • IR intergenic region
  • the promoters in these viruses are thus strong promoters due to the number of proteins that must be generated and for this reason were used in the design of this expression system.
  • the use of these promoters in expression of recombinant proteins is a novel concept for the present invention.
  • Bacteriophage PfI was selected for its ability to infect Pseudomonas species but one of ordinary skill would recognize that Pf3, fd, M13 or Xfl/Ifl and other bacteriophage promoters may also be used.
  • Two promoters derived from bacteriophage PfI were prepared, one from the PfI intergenic region (IR) and one that drives expression of the Gene-8 protein (G8).
  • a third synthetic promoter was the consensus promoter designed based on the consensus sequence for RNA polymerase which was computer designed.
  • the consensus promoter was produced by annealing SEQ ID NO: 1 and SEQ ID NO: 2 that were chosen to provide post-annealing Pstl and Xbal restriction site overhangs. Ten microliters (20 pmol) of each of the consensus promoter oligonucleotide stock solutions were mixed in a 1.5 mL microfuge tube. The tube was placed into a 95° C water bath for 5 minutes. A 250 mL beaker was filled with the 95° C water and the tube added. The beaker containing the tubes with the consensus oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 was then allowed to cool to room temperature.
  • the annealed oligonucleotides comprising SEQ ID NO: 1 and SEQ ID NO: 2 were then directly ligated into the linearized p519gfp plasmid at the Pstl/Xbal sites as shown in FIG. 3B.
  • the Pstl/Xbal sites were also used for ligation of the G8 and IR promoters, shown in FIG. 3 A and FIG. 3 C, respectively.
  • Promoters from the Gene-8 protein (G8) and intergenic region (IR) were prepared using standard PCR methods as described in Sambrook and Russell, 2001 and well known to one of ordinary skill.
  • the promoter region for the G8 coat protein of bacteriophage PfI was produced via PCR using primers set forth in SEQ ID NO: 3 and SEQ ID NO: 4, as shown in FIG. 4.
  • the G8 promoter sequence is set forth in SEQ ID NO: 5.
  • the IR promoter region, set forth in SEQ ID NO: 6 was also produced via PCR primers set forth in SEQ ID NO: 7 and SEQ ID NO: 8.
  • AU oligonucleotides were obtained from Operon Biotechnologies, Inc. (2705 Artie Street Bldg. 400, Ste. 27 Huntsville, AL 35805). All oligonucleotides were obtained in a lyophilized state and rehydrated in sterile water to a concentration of 200 pmole/ ⁇ L and a working concentration of 20 pmole/ ⁇ L was used for all reactions.
  • the promoter PCR reactions consisted of 25 ⁇ L of 2X GC buffer I (TaKaRa Minis Bio Inc.
  • G8 and IR promoters were amplified via PCR. Multiple reactions were set up with varying concentrations of template DNA. The PCR reactions were analyzed by agarose gel electrophoresis as shown in FIG. 5. PCR products at 202 bp and 240 bp correspond to the G8 and IR promoters, respectively. The two respective bands were excised and purified using a Qiagen Gel Extraction kit (Qiagen, Inc., Valencia, CA). The PCR products were then digested with Xbal and Pstl to prepare them for insertion into the p519gfp plasmid. A separate digest was run for each promoter PCR product.
  • Each digest was incubated overnight in a 37 0 C water bath, run on an agarose gel and extracted as before. As shown in FIGS. 3A-3C, the prepared promoters were then ligated into the previously double-digested p519gfp plasmid using T4 DNA ligase (Promega, Corp.)- All promoters were ligated in separate reactions.
  • Each ligation mix was transformed into E. coli One-ShotTM (Invitrogen Corp., 1600 Faraday Avenue, P.O. Box 6482, Carlsbad, California 92008) competent cells. The recovered transformation mix was plated onto LB/Kan (50 ⁇ g/mL) at a rate of 10 ⁇ L, 25 ⁇ L, and 50 ⁇ L. The plates were incubated overnight at 37° C. Colonies were selected and grown overnight in LB/Kan (50 ⁇ g/mL) broth. Kanamycin- resistant cells were used for promoter evaluation.
  • BioA is found in the reverse direction 5-prime to the bioBFHCD cluster in P. aeruginosa. Construction of complete Biotin cassette for both in vitro and in vivo overexpression of biotin related enzymes
  • a novel feature of the present invention is the redirection of the bioA gene necessary for biotin synthesis into the same cassette adjoining genes bioB, bioF, bioH, bioC, and bioD.
  • the restriction map of the complete bioABFHCD cassette is shown in FIG. 6.
  • This amplified product was consistent with the bioBCDHF cluster as the primary cassette with the final addition of the bio A gene moved to the same direction having a separate promoter but same operator, which is normal in the reverse direction.
  • Sequencing and clustering analysis using Lasergene Software (DNASTAR, Lac. ,1228 S. Park St., Madison, WI 53715) of the amplified cassette product reveals unique DNA provided in SEQ ID NO: 9 created by the novel arrangement of the necessary biotin genes and promoter constructs.
  • the biotin gene cluster cassette was amplified by PCR using primers shown in SEQ ID NO: 10 and SEQ ID NO: 11.
  • the PCR reaction used P. aeruginosa as template genomic DNA set forth in SEQ ID NO: 12 derived from either 1 ⁇ L of overnight culture of the P. aeruginosa or from a single colony picked and introduced directly into the PCR reaction tube.
  • Reactions consisted of either: a positive control reaction containing the following reagents supplied from TaKaRa Biologicals comprising 25 ⁇ L of 2X GC buffer, 8 ⁇ L of dNTP mix, 20 pmol of each control primer GCl and GC2, TaKaRa Taq, control template, and water to bring final volume to 50 ⁇ L; or, the same reaction mixture containing a single colony of P. aeruginosa and primers shown in SEQ ID NO: 10 and SEQ ID NO: 11; or, one microliter of a glycerol stock of P. aeruginosa with primers shown in SEQ ID NO: 10 and SEQ ID NO: 11.
  • Amplification conditions for each reaction were 1 minute at 94° C, 30 cycles of 94° C for 30 seconds, 51° C for 30 seconds, and 72° C for 2 minutes followed by 10 minutes at 72° C. A 4° C hold was used as needed at the end of the cycle.
  • the isolated 4.9 Kb bioBFHCD DNA fragment was annealed into the linearized pET-30/LIC vector (5.4 Kb) using kit protocols (EMD Biosciences, Inc./ Novagen, Inc., P.O. Box 12087, La Jolla, CA 92039-2087).
  • kit protocols EMD Biosciences, Inc./ Novagen, Inc., P.O. Box 12087, La Jolla, CA 92039-2087.
  • the 10.3 Kb ⁇ ET-30/EK/LIC-bioBFHCD plasmid was then transformed into One-ShotTM E. coli (Invitrogen, Corp.) resulting in 96 colonies. From these colonies, 64 were picked, grown and the plasmid DNA was isolated.
  • the plasmid DNA from each colony was digested with EcoRl to verify the size of the insert.
  • Nineteen colonies containing the correct insert were selected and subsequently digested each with EcoRl and Xbal restriction endonucleases (Promega, Corp.), and double-digested with both EcoRl and Xbal to verify proper insert dropout.
  • FIG. 9 shows the expected insert dropout for colonies 14 and 15.
  • the plasmid DNA was purified using a Qiagen Quickprep Spin Column kit. Digests were run on a 1% agarose gel. As shown in FIG. 9, 5.4 Kb and 4.9 Kb bands (lane 9) were observed that correspond to the expected size of the vector (5.4 Kb) and to the bioBFHCD cassette insert (4.87 Kb). Colony 15 displayed optimal banding and was chosen for further study.
  • Colony 15 expression vector/cassette DNA was transformed into competent One ShotTM E. coli (Invitrogen, Corp.) and plated as described previously. Five colonies were selected and plasmid DNA isolated. The DNA from each of the five colonies was digested with EcoRl or Xbal, or double-digested with both to test for DNA integrity. All colony DNA exhibited banding identical to the original DNA of colony 15 as shown in FIG. 10 for colonies one through three. DNAs for each of the five colonies were transformed into BLR(DE3) Rec A " E. coli (Invitrogen, Corp.). Cultures were grown, induced and run on a 12% SDS PAGE gel to test for the presence of proteins that would correspond to the sizes of enzymes in the biotin pathway.
  • BioA in P. aeruginosa is adjacent to the 5-prime end of bioB but in opposite orientation as shown in FIG. 1 IA. Additionally, the bioA gene has a separate promoter (PL) within the same operator region to that of the bio operon (P r ) naturally found in P. aeruginosa (Accession No. NC_002516) but which drives the expression of the gene running in the opposite direction.
  • the bioA gene was inverted and juxtaposed upstream from bioB, as shown in FIG. 6, FIG. 1 IA, FIG. 11C, and FIG. 12, to permit all of the genes in the complete cassette (bioABFHCD) to be driven by the same or perhaps multiple promoters, thus creating a synthetic biotin operon.
  • the bioA insert was produced via PCR reaction using P. aeruginosa DNA as a template, set forth in SEQ ID NO: 13, using the same protocol as described above in amplifying the bioBFHCD cassette with the exception that gene specific primers shown in SEQ ID NO: 14 and SEQ ID NO: 15 for bioA were used consisting of novel ribosomal binding sites inserted 5-prime to the bio A gene (shown in FIG. 1 IA) as well as to bioB using primers shown in SEQ ID NO: 11 (shown in FIG. 8A) and SEQ ID NO: 14.
  • the PCR cycle was similar as described before for other reactions with the exception that the annealing temperature for the primers shown in SEQ ID NO: 14 and SEQ ID NO: 15 with the P. aeruginosa bioA template was 65° C.
  • the PCR reaction produced a unique band of 1.5 Kb, shown in FIG. 14, that corresponds to the bioA gene.
  • the 1.5 Kb product was inserted into the pCRII-TOPO vector (Invitrogen, Corp.) according to the protocol provided except 1.0 ⁇ L of T4 ligase was added.
  • the pCRII/TOPO/bioA vector, shown in FIG. 11C and FIG. 12B is 5.5 Kb.
  • the ligation mix was transformed into competent E.coli One ShotTM cells as before but using 4 ⁇ L of ligation mix. E.
  • coli cells carrying this Pseudomonas- ⁇ e ⁇ ved bioA were plated onto LB agar plates containing either 50 ⁇ g/mL Kan or 50 ⁇ g/mL Ampicillin (amp) at 10 ⁇ L, 25 ⁇ L, and 50 ⁇ L.
  • Ampicillin (amp) at 10 ⁇ L, 25 ⁇ L, and 50 ⁇ L.
  • Six colonies from the Amp-selected and six from the Kan-selected colonies were picked and grown overnight at 37° C in LB plus appropriate antibiotic at 50 ⁇ g/mL.
  • the plasmid DNA from each of the picked E. coli colonies was purified using Qiagen Quick Spin protocols as before. Ndel, Xbal and double digests were run to test for the presence of the 1.5 Kb bioA dropout.
  • FIGS. 12A-12D show the insertion of the bioA sequence (FIG. 12C), excised from the pCRII- TOPO-bioA plasmid (FIG. 12B), in the same orientation and upstream of the 4.9 Kb bioBFHCD cassette annealed in the 10.3 Kb pET-30/LIC vector (FIG. 12A) to fo ⁇ n the 11.7 Kb pET- 30/LIC-bioABFHCD plasmid shown in FIG. 12D.
  • the 3ofl5 DNA was transformed into competent and inducible BLR cells for expression. Four colonies were grown and induced with IPTG as commonly known to one of ordinary skill in the art. See, e.g., Sambrook and Russell, 2001. Cells were prepared and ran on 15% SDS- PAGE gels. Controls were BLR cells without 3ofl5 DNA. The cell lysate was ran as induced and uninduced as before with IPTG. As shown in FIG. 17, these gels showed protein-banding corresponding to the sizes expected for the BFHCD enzymes based upon the P. aeruginosa sequence (Accession No. NC_002516) indicating these E. coli cells produced the P. aeruginosa biotin biosynthesis enzymes. The molecular weight for each enzyme was predicted and are provided in Table 1.
  • Each ligation was transformed into One ShotTM competent cells and plated. Several ligations and transformations were completed to generate enough colonies to go forward with screening. Each colony was picked and grown overnight in LB containing 50 ⁇ g/mL Kan. Colonies were designated 3Kl through 3K60, 4Kl through 4K29, and 5Kl through 5K24.
  • the plasmid DNA was purified as before using Qiagen Quickspin kit protocol. In each case, digests of the colony plasmid DNAs were run using Xbal, Ndel, and Xbal/Ndel as described above for bioA. As shown in FIG. 16 (lane 13), colony 3K52 showed the proper banding and it was chosen for further study.
  • DNA from 3K52 was transformed into E. coli and each of the p519-promoter plasmids for each of the PfI IR, G8 and consensus promoter DNAs was transformed into the P. mutabilis mutant host (1F9).
  • Cells were plated onto LB agar/ Kan plates for selection. The plates were incubated at 37° C overnight. Colonies were picked, grown overnight, and plasmid DNA was purified as before.
  • the promoter plasmids G8, IR, and consensus sequence were digested with EcoRl while the 3K52 DNA was digested with BamHl restriction endonuclease.
  • Each of the recesses formed were filled in with Klenow fragment (Promega, Corp.) for subsequent blunt end ligation of the two incompatible ends.
  • Klenow fragment Promega, Corp.
  • the reaction was allowed to proceed for 10 minutes at room temperature then incubated at 75° C for 10 minutes to heat inactivate the Klenow fragment.
  • 5 ⁇ L of Xbal was then added to each of the blunt end fragments.
  • the digestion reaction was allowed to react overnight at 37° C. These reactions were gel purified as before.
  • the 3K52 DNA was ligated with each of the prepared promoter DNAs to form three separate DNAs that each consisted of a unique promoter and the complete biotin cassette. Each ligation mixture was incubated at room temperature for 4 hours. The final construct is shown in FIG. 18.
  • FIG. 19 shows that 1F9 mutant cells produced DNA fragments corresponding to the correct sizes predicted for IR2-2 DNA digested with EcoRl and BamHl. The recovered cells were plated onto LB agar/ Kan.
  • FIG. 20 shows that transformed P. mutabilis 1F9 host cells produce proteins that correspond to the molecular weights of biotin operon gene products.
  • FIG. 20 is a photograph of a 12% SDS-PAGE gel showing production of recombinant biotin synthesis enzymes coded by the IR2-2 cassette expressed in the 1F9 P. mutabilis mutants. Mutated P. mutabilis cells were grown for 24 hours and 48 hours in production medium.
  • FIG. 19 shows the plasmid map of the IR2-2 DNA used for the current production of biotin.
  • the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit, or the enforceable life of any patent which may issue disclosing the cultures plus five years after the last request for a sample from the deposit.
  • the depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. AU restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
  • the culture for mutant P. mutabilis transformed with the bioABFHCD construct (lF9/pCYIR2-2) has been deposited at the American Type Culture Collection (ATCC) located at 10801 University Boulevard., Manassas, Va. 20110-2209, U.S.A. and assigned number PTA-6904.
  • ATCC American Type Culture Collection
  • the biotin synthase enzyme is coupled to a flavodoxin reductase enzyme which converts NADPH to NADP. Oxidation of NADPH is consistent with a decrease in light absorbance at 340 nm and reflects biotin synthase activity and biotin production.
  • a reaction mix was prepared consisting of 50 ⁇ iM TRIS, pH 8.0, 2 rnM DTT, 0.5 niM Fe-gluconate, 0.6 mM NADPH, 0.25 mM S-adenosylmethione, 5 mM fructose 1,6-bis phosphate, 0.5 mM L-cysteine, 0.5 mM alanine and 1 mM thiamine pyrophosphate and 0.5 mM pimelic acid.
  • the absorbance at 340 nm was monitored detecting the conversion of NADPH to NADP+ by biotin synthase.
  • SAMe is a methyl carrier essential for biotin production at two key steps: (1) conversion of 8-amino-7-oxopelargonate to 7,8-Diaminopelargonate by DAPA synthase (Ploux et al. 1999, Webster et al. 2000, Marquet et al., 2001) and (2) as a cofactor of biotin synthase enzyme in the conversion of dethiobiotin to biotin.
  • FIG. 21 is a spectrophotograph that demonstrates biotin production via the DACA assay by mutant P. mutabilis transformed with the pCYIR2-2 plasmid.
  • Cells were grown for 24 hours, harvested by centrifugation at 4° C at 6000xg for 30 minutes. The cell pellet was lysed by freeze-thawing three times followed by sonication in 30 second pulses. The cell debris was removed by centrifugation at 14000xg for 10 minutes at 4° C. The total protein of the supernatant was determined using the assay of Bradford (1976). Bradford reagent was obtained from BioRad (Hercules, CA) 100 ⁇ g of total protein was used per reaction.
  • substrate mix was added consisting of 50 mM Tris pH 8.0, 5mM MgC12, 2 mM DTT, 0.5 mM FeC12 , 0.6 mM NADPH, 0.25 mM SAM, 0.5 mM L-Cys, 0.1 mM Thiamine, 0.5 mM L-AIa, and 0.5 mM Pimelic acid.
  • FIG. 22 shows expression of the bioBFHCD cassette in the 3ofl5 plasmid in E. coli induced with IPTG.
  • Synthesis of biotin from incubation of the lysate with the above reaction mix was 1.8 ⁇ moles/hr/mg biotin synthase based on the biotin synthase enzyme representing 2% of the total protein.
  • Purification of the biotin cassette gene products, such as enzymes, may be done through a combination of ammonium sulfate precipitation and anion exchange chromatography as well as other techniques known to one of ordinary skill in the art.
  • Enzymes may be used in batch to synthesize biotin in vitro as well as each of the enzymes can be linked, for example, to a sepharose stationary column through crosslinking to CnBr activated Sepharose (Amersham-Pharmacia, Coip.) to provide an enzyme-linked synthesis for each intermediate compound.
  • CnBr activated Sepharose Amersham-Pharmacia, Coip.
  • the enzymes may be used individually to enhance or produce intermediate precursors for biotin.
  • any one or all of the following compounds can be made with the expressed compounds according to the teachings of the present invention: 8-Amino-7-oxopelargonic Acid, 7-Keto-8-aminopelargonic acid (BCAPA), 7,8-Diaminopelargonic acid (DAPA), Dethiobiotin (DTB), biotin and pimeloyl- CoA.
  • DACA 4-dimetliylaminocinnam-aldehyde
  • DACA 4-dimetliylaminocinnam-aldehyde
  • a 0.2% w/v DACA in 100% ethanol and a 2% v/v H 2 SO 4 in 100% ethanol stock solutions were prepared fresh.
  • sample was added with the total volume of sample being 100 ⁇ L (water was used to make up any difference in volume).
  • 100 ⁇ L of 0.2% DACA was added and mixed by vortexing.
  • One hundred microliters of the 2% sulfuric acid solution was added and the sample vortexed.
  • 700 ⁇ L of water was added and the reaction was incubated for 30 minutes.
  • samples were then either transferred to a cuvette and the absorbance measured at 533 nm or 200 ⁇ L of sample was transferred to a 96 well microtiter plate and the absorbance read at 533 nm.
  • a standard curve was prepared using biotin (Sigma-Aldrich Corp.) in concentrations ranging from 0-5 mg/mL.
  • FIG. 23 shows an example analysis of mutant 1F9 P. mutabilis (harboring the pCYIR2-2 plasmid) grown in continuous culture using the growth media shown in Example 1 through 56 days shifting temperatures from 33° C and 35° C during fermentation.
  • optimal biotin production was at 33° C with minimal air sparge (approximately 5 PSI) and minimal agitation. Air sparge and agitation were increased from day 32 through 42 resulting in less biotin being produced.
  • the initial conditions (through day 31) were minimal air sparge and agitation so as to prevent cells from settling. Increased air sparge was continued through days 43 to 56 with temperature being the only variable. Days 1 through 13, which are not shown, were grown at 30° C, minimal air sparge and agitation, with no biotin produced suggesting temperatures greater than 30° C are necessary for biotin production but optimal at 33° C.
  • Other examples include increasing or decreasing the concentrations of the individual components as well as substitutions for yeast extract and tryptone with equivalent fermentative by-products from yeast and other microorganisms.
  • Example 2 More optimal growth conditions were identified whereby transformed mutant P. mutabilis yield biotin at concentrations greater than 15 grams per liter. The more optimal growth conditions are shown in Example 2.
  • the media formulation listed in Example 2 to date has resulted in the 1F9 mutant P. mutabilis transformed with the pCYIR2-2 producing as much as 15 g/L of total biotin after about two weeks. Production was carried out in a lO L BFllO bioreactor (New Brunswick) at 33° C, minimal agitation and air sparge (approximately 5 PSI), with pH controlled at pH 7.8 with automatic acid (IN phosphoric acid) and base (0.5 N sodium hydroxide/0.5 N potassium hydroxide).
  • 7.5 L were grown in a batch environment and at day 14, 4 L of culture was harvested. Cells were centrifuged away and resulting supernatant was assayed directly using the DACA assay as described above. A 1/10 dilution was made of the media into water to eliminate contributions due to media with 10 ⁇ L, 25 ⁇ L and 100 ⁇ L of the 1/10 being assayed along with a standard curve of d-Biotin (Sigma) ranging from 0 ⁇ g to 500 ⁇ g. Results indicated 15 g/L of total biotin being produced in the fe ⁇ nentor.
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WO2019012058A1 (en) * 2017-07-14 2019-01-17 Biosyntia Aps CELL FACTORY HAVING IMPROVED DISTRIBUTION OF IRON SULFUR AMAS
WO2021254927A1 (en) * 2020-06-18 2021-12-23 Biosyntia Aps Methods for producing biotin in genetically modified microorganisms

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JP2020527359A (ja) * 2017-07-14 2020-09-10 バイオシンティア アペィエスＢｉｏｓｙｎｔｉａ Ａｐｓ 鉄硫黄クラスタの輸送が改善された細胞工場
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