WO2000077181A2 - Dna manipulation methods, applications for synthetic enzymes and use for polyketide production - Google Patents
Dna manipulation methods, applications for synthetic enzymes and use for polyketide production Download PDFInfo
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Definitions
- PKSs include examples of both type I (multifunctional enzyme) and type II (dissociable complex) organisation.
- Taxol The isolation of the genes coding for the proteins that make the highly potent anti-cancer compound Taxol, has not as yet been reported.
- the resulting choice for obtaining Taxol is either to cut down 200 Pacific Yew trees to obtain enough taxol for one chemotherapy session, or to make the drug chemically using one of the many exceedingly expensive and long chemical routes that have appeared recently in the literature.
- the relocation of the thioesterase domain at the end of DEBS1 was the first example demonstrating the efficacy of repositioning domains in type I modular systems. Since then, numerous such experiments have been carried out in order to probe further the efficacy of these multienzymes.
- the TE domain has been relocated at the end of module 5 as well as module 3 respectively (Kao et al., 1995, 1996). In both cases, the predicted compounds were produced that resulted from truncation of the progressing polyketide chain. Release of the 12-membered product in the former case showed that the thioesterase domain can indeed catalyse ring closure even for less energetically favourable reactions.
- two products were produced, one of them thought to be resulting from spontaneous decarboxylation.
- the first example of a chimaeric polyketide synthase constructed from a domain taken from a second PKS was demonstrated by Oliynyk ef al. (1996).
- An acyltransferase domain (AT) from module 2 of the rapamycin polyketide synthase was used to replace the AT of module 1 in the DEBS1- TE system.
- the resulting triketide lactone had a methyl group missing at position 5 of the six-membered ring. This was expected since the AT of module 2 of rap PKS (unlike the AT of module 1 of DEBS1 ) incorporates a malonyl-CoA extender unit, instead of a methylmalonyl-CoA unit.
- PKS polyketide synthases
- PKS type i polyketide synthases
- the invention provides a method of assembling several DNA units in sequence in a DNA construct.
- This method comprises the steps of: a) providing each DNA unit with a restriction enzyme recognition sequence at it's 5' end and with a recognition sequence for the same restriction enzyme at its 3' end that is combined with a recognition site for a DNA modification enzyme, b) providing a starting DNA construct having an accessible restriction site for the same or a compatible restriction enzyme and cleaving the starting DNA construct with such a restriction enzyme, c) inserting the desired DNA unit and bringing the ligated product into contact with a DNA modification enzyme such that the restriction site at the 3' end of the inserted DNA unit is abolished, d) cleaving the ligated product at an accessible unmodified recognition site for the same or a compatible restriction enzyme, e) repeating steps c) and d) to introduce each desired DNA unit to give a DNA construct containing all the desired units in sequence.
- DNA units can be any desired DNA sequence, though usually they encode enzyme domains or modules of two or more enzyme domains.
- the recognition sequences are usually positioned at the ends of the DNA unit once the DNA unit has been cut with the relevant enzyme, by this it is meant that the recognition sequences are adjacent to the coding sequence, or that they flank the said sequence.
- An accessible rest ⁇ ction site is herein defined as a restriction site which is unmodified, such that it can be cleaved by a restriction enzyme that normally recognises the sequence of the site.
- the accessible restriction site is preferably a unique site in the DNA unit or ligated product.
- the DNA modification enzyme employed in the method can be a methylase for example the dam methylase of Esche chia coli. Other methylases such as dcm are also envisaged.
- a particular method comprises the steps of a) providing each DNA unit with an Xba ⁇ recognition sequence
- 5'XXTCTAGA3' (where XX is not GA) at it's 5' end and with an Xba ⁇ recognition sequence 5'GATCTAGA3' at its 3' end.
- the recognition sequences for the restriction enzyme and the DNA modification enzyme employed in the method can be created in the DNA units prior to cutting with the restriction enzyme, for example by means of a primer extension reaction.
- the preferred DNA construct made by the method can be an expression vector capable of facilitating expression of the protein encoded by the desired DNA units.
- DNA modification can be removed and the restriction site re-established by replicating the ligated product in a dam- strain of E. coli by means of suitable vectors as known in the art.
- the invention also encompasses DNA unit assemblies where any given restriction enzyme recognition site can be modified by addition of a certain combination of nucleotide bases in order for it to be protected.
- the invention provides a method of making an assembly of several DNA units in sequence which method comprises the steps of: a) providing a first DNA unit with a recognition sequence for a first restriction enzyme at its 3' end, and cleaving the said first DNA unit with said first restriction enzyme, b) providing each other DNA unit with a recognition sequence at its 5' end for a second rest ⁇ ction enzyme which has a compatible ligation sequence with that of the first restriction enzyme, and an upstream recognition sequence for said first restriction enzyme and a downstream recognition sequence for a third restriction enzyme at its 3' end, and cleaving each said other DNA unit with the second and third restriction enzymes, c) ligating the said first DNA unit with a desired other DNA unit to form a ligated product such that the ligation of the two units abolishes the recognition site for the first restriction enzyme at the ligation junction, and cleaving the ligated product with said first restriction enzyme, d) ligating the product from c) with a desired DNA unit from b) to
- a particular method comprises the steps of: a) providing a first DNA unit with an Xbal recognition sequence
- the assembly can occur via stepwise addition of fragments to a vector.
- the first DNA unit can be attached to the solid phase for use in step c). This permits the solid phase to be split and mixed between steps c), d), and e) to make several different assemblies.
- Methods of attaching DNA units to the solid phase are well know in the art.
- Preferred solid phase elements are beads attached to the DNA units via a biotinylated nucleotide, as known in the art.
- the recognition sequences in one or more of the DNA units are preferably introduced by means of extension primers, as known in the art, though other methods such as the ligation of the required sequences or in vitro mutagenesis can also be employed.
- the assembly of several DNA units can be inserted into an expression vector and thus used to transform a host capable of expressing the protein encoded by the insert of the vector.
- the method is particularly useful where one or more of the DNA units encodes a catalytic or transport protein domain for example a ketoreductase domain from a PKS enzyme or an ACP domain from a hybrid poiyketide/peptide synthesising enzyme.
- a catalytic or transport protein domain for example a ketoreductase domain from a PKS enzyme or an ACP domain from a hybrid poiyketide/peptide synthesising enzyme.
- Such domains can be derived from enzyme domain DNA sequences from, for example, polyketide synthesising enzymes, peptide synthesising enzymes, hybrid peptide polyketide synthesising enzymes, fatty acid synthesising enzymes or other enzyme domains known in the art.
- the DNA units used in the methods of the invention can encode modules comprising one or more catalytic or transport domains. Usually a module contains all of the domains required to complete one condensation step in the synthesis of a target molecule.
- Alternative aspects of the invention resulting from the methods of the invention include: DNA constructs or vectors incorporating a DNA assembly encoding synthetic enzymes, synthetic enzymes encoded by such DNA assemblies, hosts expressing synthetic enzymes, hybrids of transformed hosts expressing synthetic enzymes, and compounds produced by the synthetic enzymes. Where the product produced by the synthetic enzyme exhibits toxicity to a host stain, this can be worked around e.g. by means of choosing a different strain or mutating the original strain to provide mutants which are more tolerant.
- the diversity of compounds produced by hosts transformed with the synthetic enzymes of the invention can be further increased by using known methods of using different feedstocks in the fermentation to provide different starter units for the desired product. Where yield of desired synthetic enzyme product is low, routine steps e.g. mutation and selection, can be taken to improve this,
- the synthetic enzymes of the invention can also be used in cell-free systems to produce the desired target molecule in vitro as known in the art, for example, see Carreras and Khosla (1998).
- the invention provides a method of synthesising a target molecule comprising the steps of a) examining the composition and stereochemistry of a target molecule, b) determining which catalytic and transport domains need to be present in a synthetic enzyme in order to catalyse the synthesis of the target molecule, c) using any one of the methods of the invention to assemble the required DNA units encoding the catalytic and transport domains into a DNA assembly that encodes said synthetic enzyme which is capable of synthesising the target molecule. d) placing the DNA assembly into a vector to allow expression of the synthetic enzyme in a host capable of synthesising the target molecule after transformation with said vector.
- Target molecules are generally bio-active molecules, usually having a predominantly carbon based backbone and usually are macromolecules comprised of condensed units.
- the transformed host can be tested for the presence of the target molecule after step d). If yields of the desired compound are low then conventional methods of improving product yield from, for example Streptomycetes, can be employed.
- Transformed hosts which result from the methods of the invention and their use in producing target molecules are also aspects of the invention.
- Hosts suitable for transformation with the DNA assemblies of the invention are known in the art and include insect or mammalian cells, though more usually suitable are bacterial cells, for example, the improved host strains described by Ziermann and Betlach (1999).
- the synthetic enzyme can be used in a cell-free system to produce the target molecule in vitro.
- a further aspect of the invention is a method of making a synthetic enzyme to catalyse the synthesis of a target molecule comprising the steps of a) examining the composition and stereochemistry of a target molecule, b) determining which catalytic and transport domains need to be present in the synthetic enzyme in order to catalyse synthesis of the target molecule, c) using any one of the methods of the invention to assemble the required DNA units encoding the catalytic and transport domains into a DNA assembly that encodes an enzyme which is capable of synthesising the target molecule, d) expressing the DNA assembly in a suitable host to produce the enzyme.
- each DNA unit has a recognition sequence for a restriction enzyme at it's 5'-end and a second recognition sequence for the same or a compatible enzyme at it's 3'-end which incorporates a recognition sequence for a DNA modifying enzyme.
- each DNA unit has an Xbal recognition sequence 5'XXTCTAGA3' (where XX is not GA) at it's 5'-end and an Xbal recognition sequence 5'GATCTAGA3' at it's 3'-end
- each DNA unit has a recognition sequence at its 5' end for a first restriction enzyme, and a downstream recognition sequence for a second restriction enzyme followed by a downstream recognition sequence for a third restriction enzyme at its 3' end, such that the DNA units, once restricted by the first and second restriction enzymes can be ligated together to abolish the restriction sites at the ligation junction.
- each DNA unit has a Spel recognition sequence 5 ⁇ CTAGT3' at its 5'-end, and a downstream Xbal recognition sequence 5TCTAGA3' followed by a downstream Smal recognition sequence 5'CCCGGG3' at it's 3'-end
- Catalytic or transport protein domains can be derived from any enzyme, for example those listed above.
- Particularly envisaged are libraries in which the DNA units encode polyketide synthetic domains, comprising two KS domains, at least two AT domains, two KR domains, two DH domains, two ER domains, an ACP domain and a TE domain.
- modules comprising a DNA sequence encoding a functional set of polyketide synthetic domains wherein the module has a recognition sequence for a restriction enzyme at it's 5'-end and a second recognition sequence for the same or a compatible enzyme at it's 3'-end which incorporates a recognition sequence for a DNA modifying enzyme.
- An envisaged module has an Xbal recognition sequence 5'XXTCTAGA3' (where XX is not GA) at it's 5'-end and an Xbal recognition sequence 5'GATCTAGA3' at it's 3'-end
- a module comprising a DNA sequence encoding a functional set of polyketide synthetic domains can have a recognition sequence at its 5' end for a first restriction enzyme, and a downstream recognition sequence for a second restriction enzyme followed by a downstream recognition sequence for a third restriction enzyme at its 3' end, such that the DNA units, once restricted by the first and second restriction enzymes can be ligated together to abolish the restriction sites at the ligation junction.
- the module has a Spel recognition sequence 5 ⁇ CTAGT3' at its 5'-end, and an upstream Xbal recognition sequence 5TCTAGA3' and a downstream Smal recognition sequence 5'CCCGGG3' at it's 3'-end.
- modules wherein the DNA units encode polyketide synthetic domains, comprising two KS domains, at least two AT domains, two KR domains, two DH domains, two ER domains, an ACP domain and a TE domain. It is also envisaged that other non-polyketide enzyme domains can be included in the modules provided by the invention.
- vectors containing one or more modules are also provided. Particularly useful are vectors in which a non-functional recA gene is also present. Such vectors prevent unwanted homologous recombination occurring between domains within the vector upon integration into a suitable host by abolishing the recA gene activity in that host.
- the invention also provides a method of transforming a host with one or more synthetic DNA assemblies encoding enzyme domains which comprises the steps of: a) Inserting said DNA assembly into a vector containing a mutated internal fragment of a recA gene sequence such that the vector is capable of undergoing homologous recombination with the recA gene of the host, b) bringing said vector into contact with a host chromosome under conditions which permit homologous recombination to take place, c) disrupting the host recA gene by the integration of the DNA of said vector into the chromosome.
- the expression vector can be used to transform a Steptomyces host.
- the DNA assemblies contained in the vector can be modules as described herein. Also envisaged are transformed hosts which prior to transformation with a vector containing one or more modules according to the invention, were already lacking a recA function.
- kits containing DNA units, DNA modules, vectors, DNA manipulation hosts, DNA modification hosts, expression hosts, or solid phase elements for use in the methods of the invention.
- a kit might contain a first DNA unit which is a vector suitable for transforming a suitable host, a library of modules for insertion into that vector, both the first DNA unit and the library having the necessary recognition sites for use in the methods of the invention, together with host strains suitable for the manipulation and expression of the DNA assemblies of the invention.
- a de novo "domain-by-domain" reconstruction of a hybrid multienzyme from the erythromycin-producing PKS has been achieved by the inventors by assembling DNA units corresponding to the constituent domains. The assembled gene was expressed in S. erythraea and the expected compounds were isolated from the bacterial broth.
- Application of this methodology, or variations of this methodology for making combinatorial assemblies of complex and aromatic PKSs allows for the rapid generation of novel or altered PKS or other synthetic multienzymes and paves the way for a quick and inexpensive synthesis of potentially bio- active molecules.
- One alternative to chemical syntheses is to carry out a 'retrobiosynthetic analysis' of the desired molecule, by pinpointing the exact number and type of synthetic enzyme domains that are required for every chemical step, and then assembling the DNA units that encode these enzymes in order to make a hybrid synthetic enzyme.
- the aim is therefore, to assemble these domains or even modules in a manner as desired, so that the linked enzymes can carry out a progressive synthesis of a desired target molecule.
- flanking both ends of the DNA of the desired DNA unit (domain or module) with a recognition sequence that is cleaved on one end by Xbal, and on the other end by a restriction enzyme that is compatible with Xbal (e.g. Spel) is possible.
- This strategy makes use of selective recognition of the restriction enzyme site by the restriction enzyme Xbal, depending upon the sequence adjacent to the restriction enzyme site and upon the strain used (dam + or dam " ) during the assembly process.
- the method has been shown to be successful, and by using this methodology to assemble modules, the complete erythromycin-producing PKS (comprising of six modules coded by three large open reading frames) can be built in under 10 days. Even though this time-period is small compared to what it would take to assemble the ery PKS genes using conventional methodologies, using a variation of the above mentioned methodology, complete gene-clusters, like the 33 kbp erythromycin PKS, can be built within a matter of hours.
- the methodology thus outlined requires DNA units to be modified so that they contain the appropriate 5'and 3' ends (X and X d respectively). These units are then progressively assembled to achieve the desired gene length. The vector containing the assembled or reconstructed gene is then used to transform an expression system to achieve protein expression. This methodology has been shown to work effectively - the hybrid multienzyme DEBS1-TE was reconstructed by assembling de novo the ten constituent domains. The assembled gene, when expressed in S. erythraea gave the expected six-membered triketide lactones.
- inter-modular recombination events within the reconstituted PKS or other synthetic enzyme gene may preclude the use of identical PKS or other enzyme domain DNA units in a set of modules. It might be expected that, for example ( Figure 2) the ACP * DNA in module 1 to recombine with the identical ACP * DNA in module 3. This event can take place, for example, when the expression vector that possesses the assembled gene containing numerous identical PKS DNA units is used to transform a streptomyces host for polyketide production.
- the inventors have developed a strategy that can circumvent this problem, therefore making it possible to construct large synthetic enzyme gene clusters using identical domains or modules repeatedly. This translates into a less expensive route towards synthetic enzyme gene construction (one would not require to have a start-up library of 200 or so to cover all possibilities), as the set of 12 domains, or similar functional arrangements of domains, are true "off-the-shelf components for the assembly of PKS genes or genes for other hybrid synthetic enzymes.
- the inventors provide methods of DNA assembly that pave the way for a cheap and fast synthesis of a host of bio-active molecules, e.g. the anti- cancer drug Discodermolide.
- Figure 1 shows the chemical/stereochemical choices that each PKS domain can make. A total of 12 domains are required for every conceivable polyketide reaction.
- Figure 2 shows integration of a plasmid containing more than one identical DNA unit (ACP * ). After the plasmid has integrated in the streptomyces host through homologous recombination with TE, internal recombination can occur to yield truncated PKS genes. This is because the host is recA + .
- Figures 3A and 3B show a schematic representation of the assembly process.
- the de novo construction of DEBS1-TE DNA fragments (units) encoding for the constituent domains of the multienzyme DEBS1-TE were inserted sequentially into the expression plasmid pCJR24.
- the final plasmid pAR10 was then expressed in S. erythraea/JC2 to yield the expected triketide lactone products that are synthesised by the schematically shown re-assembled DEBS1-TE synthase.
- the amino acid changes made within the linker regions between domains are shown below the actual amino acid sequence.
- Figure 4 shows the methodology of the assembly of DNA units using Xbal/dam methylase technology.
- transformation of a Dam ' strain with plasmid (as it is a dam ' strain, even X d would be cleaved by Xbal) is effected.
- Cutting is achieved by Xbal and the DNA unit purified on a gel.
- Figure 5 shows the procedure for the assembly of DNA units using Xbal/dam methylase technology.
- Figure 6 shows how an Xbal site can be made sensitive to methylation.
- the RE cuts at the sites shown by arrows.
- the boxed sequence is methylated in a dam + strain thereby altering the Xbal recognition site.
- the sequence however is not methylated in a dam strain, and so can still be cleaved by Xbal.
- the Xbal recognition sequence (5 CTAGA3') can therefore be selectively cleaved by Xbal. Assembly of DNA units uses only one restriction enzyme - Xbal.
- Figure 7 shows the methodology of the in vitro assembly of DNA units - I using solid phase beads with the enzymes Xbal, Spel and Smal (other Xbal - compatible REs may be used).
- Figures 8 and 9 show how the methodology of the in vitro assembly of DNA units - II would proceed to the point of placing the DNA assembly into an expression vector for transforming and appropriate host.
- Figure 10 shows how in one single ligation, 16 ongoing assemblies are generated. This cascade can obtain exponential proportions.
- the gene library can be increased by increasing the diversity of the incoming unit.
- Figure 11 shows the integration of an expression plasmid into a streptomyces host, using a mutated internal fragment of the recA gene as the region for homologous recombination.
- the resulting PKS gene can now contain more than one identical DNA units as the strain has been made recA minus.
- Figure 12 shows the assembled PKS recADEBS1-TE.
- the second module is composed of domains that normally belong to the first module.
- Figures 14A and 14B show a DNA sequence alignment of the recA gene S. lividans (S.I) and S. ambofaciens (S.a). Start of the gene is from 'ATG' and stop is TGA'. Percent similarity: 94.713, percent identity: 94.713.
- Figure 15 shows how an Xbal/Spel system might be used instead of an Xbal/dam methylase system to assemble DNA units, a strategy involving compatible restriction enzymes flanking either end of a DNA unit.
- An example of compatible REs would be Xbal and Spel.
- the recognition sequence of Xbal is - 5TCTAGA3' and that for Spel is 5 ⁇ CTAGT3'. After Xbal and Spel have cleaved the DNA at their respective sites, the DNA unit can be ligated together as the overhanging is complementary. The junction where any two units are joined is now recognised by either Xbal or Spel.
- Figure 16 is a schematic representation of the compatibility of Xbal- and Spel- digested DNA overhangs. It shows the compatibility of the sticky ends produced by Xbal and Spel and how re-ligation abolishes both sites.
- Figure 17 shows a schematic representation of the erythromycin-producing polyketide synthase; primary organisation of the genes and their corresponding protein domains.
- the multienzymes deoxyerythronolide B synthase 1 (DEBS1), DEBS2 and DEBS3 each have two modules, each of which processes one cycle of polyketide chain extension. Each of the six modules is constituted by covalently-linked enzymatic domains. Exploitation of such an enzymatic hierarchy as "of-the-shelf reagents can lead to synthesis of important chemical compounds.
- Figure 18 shows the structure of the anticancer drug discodermolide (top) and the 'retrobiosynthetic approach' towards synthesising a target molecule (a discodermolide).
- a discodermolide a target molecule
- Such an approach would involve opening up the structure (a.), identifying the number and type of polyketide carbon units that would make the discodermolide carbon skeleton (b.), and choosing the PKS DNA units (modules/domains) responsible for the uptake and subsequent processing of the carbon units (c).
- Figure 19 shows the anti-tumour compound octalactin and the strategy behind the retrobiosynthetic approach towards synthesising bio-active molecules.
- the strategy comprises the steps of: 21a
- Figure 20 shows a schematic representation of they hypothetical polyketide synthase for synthesising octalactin B, assembled from enzyme units that belong to various PKSs in the public domain.
- Figure 21 shows a schematic representation of the hypothetical decarestrictine polyketide synthase for synthesising the anti-cholesterol compound decarestrictine J, assembled from enzyme units that belong to various PKSs in the public domain.
- Example 1 Vectorial assembly of DNA units
- DNA units that are to be assembled contain the Xbal recognition sequence at either end of the unit.
- two nucleotides are arranged at the 5' end of the Xbal recognition sequence (thus making it 5'GATCTAGA3'). This is achieved by first incorporating the Xbal recognition sequences in the oligonucleotide primers and then amplifying the desired DNA unit by PCR. The PCR products are then ligated to a pUC-18 vector, used to transform a dam + strain of E. coli, and the clones isolated and sequenced for possible errors in the PCR products. A dam + strain of E.
- coli- like DH10BTM - methylate the nucleotide A in the sequence GATCTAGA, as 5'GATC3' is a sequence that is recognised by the product of the Dam methylase gene (Fujimoto ef a/.,1965; Geier et al., 1979). This makes only one end of the DNA unit cleavable by Xbal.
- the vector is then used to transform a dam " strain of E. coli (e.g. ET12567 - MacNeil et al. (1992)) and the plasmid DNA isolated. This DNA is now cleavable at bofb ends of the DNA unit by Xbal.
- DEBS1-TE a multienzyme that has the first of the three bimodular erythromycin DEBS enzymes (DEBS1), fused with the erythromycin thioesterase (Cortes et al., 1995) was constructed in a de novo fashion.
- DEBS1-TE a multienzyme that has the first of the three bimodular erythromycin DEBS enzymes (DEBS1), fused with the erythromycin thioesterase (Cortes et al., 1995) was constructed in a de novo fashion.
- the ten inherent PKS domains in DEBS1-TE namely, loading module (itself composed of an AT and an ACP), KS1 (ketosynthase of module 1), AT1 , KR1 , ACP1 , KS2 - 23 -
- the DNA for all ten domains was amplified by PCR to incorporate the two aforementioned recognition sequences for Xbal (5TCTAGA3' and 5'GATCTAGA3') at the 5' and 3' ends of the DNA unit respectively.
- the PCR products were cloned in pUC18 vector, sequenced, and then used to transform the dam " E. co// ET12567 strain.
- the DNA unit for TE was inserted into S. erythraea expression vector pCJR24 (Rowe et al., 1998) which has a unique Xbal site. This vector also contains a thiostrepton-resistance gene as a marker for identifying successful integrands.
- the ligated products were used to transform the dam + E. co// DH10BTM strain and the plasmid DNA isolated.
- This plasmid (pAR1) can only be singly cleaved with Xbal, despite possessing two Xbal recognition sequences, as one of the sites (situated at the 3' end of the TE unit) has been methylated by the E. coli Dam methylase.
- the next DNA unit (ACP2 from module 2 of DEBS1) was then ligated to the Xbal-cut pAR1 , the ligation mixture used to transform DH10B cells and the plasmid DNA isolated.
- Plasmid pAR10 was then used to transform S. erythraea/JC2 - a mutant strain of the wild- type S. erythraea NRRL2338 that lacks the DEBS genes except for the TE DNA fragment (Rowe et al., 1998).
- Thiostrepton-resistant colonies were selected upon integration of the vector into the S. erythraea chromosome. Single transformants were grown on selective media, as described in the methods section. The fermentation broth was extracted with ethyl acetate - 24 -
- E. coli dam + DH10BTM strain was purchased from Gibco BRL, USA.
- Pfu DNA polymerase was purchased from Boeringer, Germany. Construction of the final expression plasmid pAR10 was carried out in several steps, as follows. The ten PKS DNA units were amplified by PCR using pfu DNA polymerase. The respective regions of eryAI gene, as well as the oligonucleotides used for each PCR are outlined: LM - segment of ety /gene (Bevitt et al., 1992) extending from nucleotide (N) 588 to N 2389;
- KR1 - segment of eryAI gene extending from N 4808 to N 6316; 5'GGTCTAGAGTCGGTGCACCTGGGCACCGGAGCACGCCGGGTGCCC
- TE - segment of eryAIII gene (Donadio et al. 1991) extending from N 8753 to N 9602; 5'GGTCTAGACAGCGGGACTCCCGCCCGGGAAGCG3' and 5'GGGCTAGCTCTAGATCATGAATTCCCTCCGCCCAGCCAGGCGTC3'. All PCR products were 5' phosphorylated and ligated to Smal-cut, dephosphorylated pUC18 vector and used to transform E. co// DH10B electrocompetent cells. The desired plasmids - containing the amplified DNA fragments were isolated and sequenced using standard pUC forward and reverse primers. No mistakes in the amplified products we.e detected.
- Plasmid pAR1 was isolated, digested with Xbal, and ligated to the ACP2 fragment, and ligation products treated as mentioned above.
- the other DNA fragments namely, KR2, AT2, KS2, ACP1 , KR1 , AT1 and KS1 were sequentially added to finally yield plasmid pAR10.
- This plasmid was then digested with ⁇ / el and Xbal restriction enzymes and ligated with the LM fragment previously digested with the same two enzymes. The ligated products were used to transform E. coli DH10B electrocompetent cells and the final expression plasmid pAR10 isolated. Plasmid pAR10 was then used to transform S. erythraea/JC2 strain and colonies carrying the expression plasmid were selected through resistance to thiostrepton upon integration of the plasmid into the S. erythraea chromosome. Single transformants were picked and grown on - 28 -
- Figure 7 outlines the strategy for the in vitro assembly of PKS DNA units.
- the inventors have constructed the multienzyme DEBS1 -TE.
- the in vivo construction of the gene for DEBS1 -TE took 12 days to complete.
- the in vitro assembly on the other hand was completed in 2 days.
- LM, KS1 , KR1 , AT1 , ACP1 , KS2, AT2, KR2, ACP2 and TE were amplified by means of PCR.
- the forward primer in all cases, except the LM contained the Spel recognition sequence 5 ⁇ CTAGT3' while the reverse primer was engineered in such a way that it contained the Xbal recognition sequence 5' TCTAGA3' and Smal recognition sequence 5'CCCGGG3' downstream of the Xbal site ( Figure 7).
- the amplification of the LM was carried out using a biotinylated forward primer and a reverse primer that contained the Xbal recognition sequence (5 CTAGA3').
- PCR products were cloned in pUC-18 vector and the resulting plasmids sequenced to detect possible errors introduced by PCR. All plasmids, except the one containing the LM unit were then digested with Spel and Smal, dephosphorylated in order to remove the 5' phosphate group and the appropriate fragments isolated and eluted.
- the LM unit was cleaved with Xbal and attached to a bead that was coated with streptavidin (following the manufacturer's instructions) as shown in figure 7.
- the assembly process was initiated by adding DNA ligase to the tube containing a large excess of the first unit (KS1) and LM-bead.
- the reason for having a large excess of the KS1 unit compared to the LM-bead unit is to favour the LM-bead ligating to the incoming unit, as opposed to the self-ligation of the LM-bead (see figure 7).
- the ligation of the two DNA fragments is unidirectional as only the Spel-cut end of KS1 complements the Xbal-cut end of the LM-bead.
- the desired product of the ligation reaction namely 'bead-LM-KS1' was separated from the reaction mixture and washed. This product was then cleaved with Xbal, in order to activate the 3' end of KS1.
- the beads were washed again to remove the small Xbal-Smal DNA fragment that was - 30 -
- a strategy employing the invention in order to construct the highly potent anti-breast cancer drug discodermolide, the anticholesterol compound decarestrictine, and the antitumour compound octalacin using polyketide synthase domains/modules is outlined below. - 31 -
- the drug discodermolide ( Figure 18), isolated from the marine sponge 'Discodermia disoluta', has been identified as a highly potent anti-cancer compound and 80 times more effective than the well known anticancer drug Taxol (TerHarr et al., 1996). It has the same mechanism of action as Taxol, even though it is structurally different from the latter.
- discodermolide is a polyketide and can therefore be constructed from a system that has the basic enzymatic building blocks (domains and modules) that make other polyketides like erythromycin and rapamycin. Having predicted that approximately 45 domains housed in 12 modules would be required in order to carry out the chemistry that accounts for the functionalities on the carbon skeleton of discodermolide, one can now begin to construct such a system. All one has to do is to identify the type and nature of the domains/modules that one requires to generate the observed functionalities, and then assemble these units in the desired order ( Figure 18). The resulting DNA assembly can then be put into a bacterial strain that makes a functional polyketide synthase.
- discodermolide can be made available through chemical synthesis - there have been a few chemical routes reported in literature recently (Marshall and Johns, 1998 and references therein). However, as is the case with most other complex molecules, large scale production of discodermolide, using the chemical route would turn out to be excessively expensive. Chemists have been using the retrosynthetic analysis approach towards total synthesis of important bioactive molecules. This approach breaks the target compound into many smaller pieces - easily synthesised - which are then re- assembled.
- the unit-DNA segments are amplified using the polymerase-chain-reaction (PCR) - from
- Suitable vectors have an antibiotic resistance marker (for selection of this vector on an antibiotic-rich media) and an "origin-of -replication" (ori). Ori is essential for
- vectors for the expression of the synthetic enzymes of the invention are the actinomycete vectors described by Rowe et al. (1998).
- the strain is then grown in a media that is supplemented with the
- Figures 4 and 5 show how the assembly proceeds.
- Octalactin A and B are natural products isolated from the marine gorgonian octocoral 'Pacifigorgia sp.' (Tapiolas et. al., 1991).
- Octalactin A shows very strong cytotoxicity toward B-16-F-10 murine melanoma and HCT-116 human colon tumour cell lines and is a promising drug candidate, while octalactine B displayed no such activity (Tapiolas et. al., 1991).
- Total syntheses of both octalactin A and B have been reported in literature. One such synthesis (Buszek, et.
- the molecule decarestrictine J can be synthesised using the retrebiosynthetic approach.
- Decarestrictine J is a ten-membered lactone that comes from the family of decarestrictines, shown to display strong anti- cholesterol activity (Grabley et. al., 1992). The total synthesis of Decarestrictine J has been reported and involves numerous chemical steps (Yamada et. al., 1995).
- the target molecule (figure 21) can be conceived to be formed by assembly of five acetate polyketide units. Using the retrobiosynthetic approach, one can identify the PKS domains/modules that - 37 -
- decarestrcitine PKS is shown in figure 21.
- the loading module, as well as the four internal modules along with the TE domains can be conveniently assembled using the invention.
- the assembled 'decarestrictine gene' can then be expressed in a suitable host in order to check for the production of decarestrictine J.
- the retrobiosynthetic approach involves the following steps; a). Identification of the number and nature of carbon units that make up the target molecule b). Identification of the modules/domains from libraries of polyketide/peptide synthetase/fatty acid/etc. encoding units that are responsible for the uptake of the said carbon units and the nature and degree of functionalisation of the carbon chain c). Assembly of the said modules/domains using the methods of the invention d). Expression of the assembled gene in a suitable expression host.
- Example 4 Transforming strains with DNA encoding similar synthetic enzyme domains
- recA E. coli strain
- the vector, into which the assembled gene is being constructed contains a portion of a streptomyces recA gene.
- This recA fragment carries a mutation.
- the vector is used to transform a streptomyces host (e.g. S. lividans or S. erythraea).
- the fragment of recA gene carrying a mutation recombines with the recA gene of the streptomyces host, abolishing the functional recA gene and making the strain recombination minus ( Figure 11).
- the 1.0 kbp recA fragment, flanked at both ends by an Xbal site was then inserted in the expression vector pCJR24 that has a unique Xbal site.
- the ligation mixture was used to transform E. co// DH10B cells and the desired plasmid DNA isolated.
- the resulting plasmid (pARecA24) contains a non- methylated Xbal site at the 5' end of the recA gene fragment.
- the ten PKS DNA units, namely, TE, two each of ACP1 , KR1 , AT1 & KS1 , and LM were inserted into the plasmid pARecA24 to finally yield the expression plasmid pfiecADITE.
- This plasmid was used to transform wild-type S. lividans protoplasts, and thiostrepton resistant colonies were grown in defined liquid media as described above.
- the compound ( Figure 12) was isolated from the bacterial broth and chemically character
- the first gene in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus codes for a unique PKS module coupled to a peptide synthetase. J. Mol. Biol. 286,465-474.
- Discodermolide a cytotoxic marine agent that stabilizes microtubules more potently than taxol. Biochemistry 35, 243-250.
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CA002376559A CA2376559A1 (en) | 1999-06-11 | 2000-06-12 | Dna manipulation methods, applications for synthetic enzymes and use for polyketide production |
AU55457/00A AU5545700A (en) | 1999-06-11 | 2000-06-12 | Dna manipulation methods and applications for synthetic enzymes |
EP00940533A EP1190045A2 (en) | 1999-06-11 | 2000-06-12 | Dna manipulation methods , applications for synthetic enzymes and use for polyketide production |
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Cited By (4)
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WO2001081564A2 (en) * | 2000-04-26 | 2001-11-01 | Actinodrug Pharmaceuticals Gmbh | Method for producing dna encoding polypeptides that are composed of several sections, and for producing polypeptides by expressing the dna thus obtained |
US8999679B2 (en) | 2008-12-18 | 2015-04-07 | Iti Scotland Limited | Method for assembly of polynucleic acid sequences |
US9777305B2 (en) | 2010-06-23 | 2017-10-03 | Iti Scotland Limited | Method for the assembly of a polynucleic acid sequence |
CN113728130A (en) * | 2019-04-01 | 2021-11-30 | 国立大学法人神户大学 | Construction method of chimeric plasmid library |
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- 1999-06-11 GB GBGB9913694.7A patent/GB9913694D0/en not_active Ceased
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- 2000-06-12 AU AU55457/00A patent/AU5545700A/en not_active Abandoned
- 2000-06-12 EP EP00940533A patent/EP1190045A2/en not_active Withdrawn
- 2000-06-12 WO PCT/GB2000/002286 patent/WO2000077181A2/en active Search and Examination
- 2000-06-12 CA CA002376559A patent/CA2376559A1/en not_active Abandoned
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001081564A2 (en) * | 2000-04-26 | 2001-11-01 | Actinodrug Pharmaceuticals Gmbh | Method for producing dna encoding polypeptides that are composed of several sections, and for producing polypeptides by expressing the dna thus obtained |
WO2001081564A3 (en) * | 2000-04-26 | 2002-04-25 | Florian Schauwecker | Method for producing DNA encoding polypeptides that are composed of several sections, and for producing polypeptides by expressing the DNA thus obtained |
US8999679B2 (en) | 2008-12-18 | 2015-04-07 | Iti Scotland Limited | Method for assembly of polynucleic acid sequences |
US9777305B2 (en) | 2010-06-23 | 2017-10-03 | Iti Scotland Limited | Method for the assembly of a polynucleic acid sequence |
CN113728130A (en) * | 2019-04-01 | 2021-11-30 | 国立大学法人神户大学 | Construction method of chimeric plasmid library |
EP3951028A4 (en) * | 2019-04-01 | 2023-01-18 | National University Corporation Kobe University | Method for constructing chimeric plasmid library |
US11643648B2 (en) | 2019-04-01 | 2023-05-09 | National University Corporation Kobe University | Method for constructing chimeric plasmid library |
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AU5545700A (en) | 2001-01-02 |
CA2376559A1 (en) | 2000-12-21 |
GB9913694D0 (en) | 1999-08-11 |
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