Patent Grant
8143035
This application is a 35 U.S.C. Section 371 of PCT/US2006/037107 filed Sep. 22, 2006, and incorporated herein by reference in its entirety.
The invention relates to the synthesis of polyketides in Actinomycetes by overexpression of pantothenate kinase (PanK) and supplementation with pantothenic acid, thus increasing in vivo CoA production and thereby also increasing the production of secondary metabolites, such as polyketides.
The Actinobacteria or Actinomycetes are a phylum of Gram-positive bacteria. Most are found in the soil, playing an important role in decomposition of organic materials such as cellulose and chitin. Other Actinobacteria colonize plants and animals, and include a few pathogens such as Mycobacterium. Representative genera include: Actinomyces, Arthrobacter, Bifidobacterium, Corynebacterium, Frankia, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, and Streptomyces.
The actinobacteria are unsurpassed in their ability to produce compounds that have pharmaceutical activity. As early as 1940, Selman Waksman discovered that the soil bacteria he was studying made the antibiotic “actinomycin” and was awarded the Nobel Prize for his work. Since then hundreds of naturally occurring antibiotics have been discovered in these terrestrial microorganisms, especially from the genus Streptomyces. Therefore, this phylum is a very important source of medicinal compounds, and there is always a need to maximize the production of such pharmaceuticals.
One way of improving the production of any compound made by bacteria is through metabolic engineering—the purposeful re-design of an organism's metabolic pathways by recombinant DNA techniques. Metabolic engineering has the potential to considerably improve process productivity and is increasingly used in both academic and industrial institutions. Most current metabolic engineering studies have focused on manipulating enzyme levels. However, cofactors also play an essential role in a large number of biochemical reactions and their manipulation has the potential to be used as an additional tool to achieve desired metabolic engineering goals.
For example, Coenzyme A (CoA) is an essential cofactor in numerous reactions and is involved in the regulation of key metabolic enzymes. In fact, it has been estimated that as much as 4% of all enzymes utilize CoA, CoA thioesters, or 4′-phosphopantetheine as substrates. Further, CoA compounds, including acetyl-CoA, propionyl-CoA, malonyl-CoA, and methylmalonyl-CoA, are essential precursors required for the biosynthesis of polyketides and other complex biomolecules.
Polyketides are small, cyclized molecules. Between 5000 and 10,000 are known, and about 1% of them possess drug activity. Sales of the more than 40 polyketide drugs—including antibiotics, immunosuppressants, cholesterol-lowering agents, antifungals, and cancer chemotherapeutics—exceed $15 billion a year. But polyketides are difficult to synthesize chemically, and the exotic microbes that produce them naturally can be hard to grow in culture.
Sugars are the structural components of different types of natural products. Important antibiotics, antifungals, antiparasites and anticancer drugs possess sugars attached to the aglycon core. These sugar components participate in the molecular recognition of the cellular target by the bioactive compound and, therefore, its presence is important, in many cases essential, for the biological activity of many natural products. A great majority of these sugars belong to the 6-deoxyhexoses (6DOHs). These sugars are synthesized from nucleoside diphosphate-activated hexoses (mainly D-glucose) via a 4-keto-6-deoxy intermediate. Two common enzymatic steps leading to the biosynthesis of this intermediate are catalyzed by a dNDP-D-hexose synthase and dNDPD-hexose-4,6-dehydratase. The different 6DOHs will vary depending on the substituents and/or the stereochemistry at carbon atoms at positions 2, 3, 4, or 5 of the hexose carbon chain, resulting from deoxygenations, transaminations and/or C, N, or O methylations. D- and L-isomeric forms of many 6DOHs exist as a result of the action of a 5- or a 3,5-epimerase.
In recent years, a number of 6DOHs gene clusters have increasingly been characterized, most of them participating in the biosynthesis of different antibiotics produced by Actinomycetes. Deoxy sugar carrying glycosides and polysaccharides are believed to be secondary gene products, which are synthesized from activated sugars by glycosyltransferases. The activation is brought about in analogy to mammalian pathways by nucleoside diphosphates. It is the activated sugar that is derivatized in general before being transferred to the respective aglycon. The deoxysugars are transferred to the corresponding aglycon by glycosyltransferases, which are generally sugar-, aglycon-, and site-specific. In recent years, increasing evidence has suggested some degree of “flexibility” of glycosyltransferases involved in the biosynthesis of secondary metabolites, and there have been some reports of examples in which different deoxysugars have been transferred by a glycosyltransferase to its aglycon. One of these glycosyltransferases, the elloramycin glycosyltransferase (ElmGT), has been shown to be especially “flexible” in accepting different L- and D-deoxysugars and also being able to transfer a disaccharide. Recently, several plasmids that direct the biosynthesis of L-daunosamine, L-Olivose, L-oleandrose or D-desosamine have been reported by the different groups. These plasmids contain different subsets of genes involved in the biosynthesis of these deoxysugars from several antibiotic-producing organisms.
What is needed in the art are metabolic engineering methods that can be applied to Actinomyces, such as Streptomyces, to increase the production of polyketides and other complex biomolecules.
E. coli have been already been metabolically engineered to increase CoA production (4), whereby a 10 fold increase in CoA and a 5 fold increase in acetyl-CoA was seen on overexpression of the panK gene and supplementation with pantothenic acid. However, the metabolic engineering of E. coli is not easily applied to the Actinomycetes, which is a much more difficult bacterial phylum to engineer (2). Further, early attempts were only made to improve the production of primary CoA products and acetyl-coA, and improved production of secondary metabolites was not attempted.
The invention generally relates to the metabolic engineering of Actinomycetes. By using the panK gene with pantothenic acid as a supplement to promote the biosynthesis of CoA in polyketide producing Actinomycetes, the levels of secondary metabolites such as polyketides production are increased. Other compounds, including amino acids, can also be optimized in the media composition to further improve yield.
R
Polyketide production in an Actinomycetes cell is increased by producing an engineered Actinomycete that overexpresses a pantothenate kinase (panK) gene, and growing the cell in a medium supplemented with pantothenic acid, this causes the bacteria to produce more polyketide than a wild type Actinomycete grown in said medium not supplemented with pantothenic acid.
An engineered Actinomycetes cell comprising a recombinant pantothenate kinase (panK) gene is also described. The engineered Actinomycetes increases PanK activity and polyketides synthesis.
Polyketide production is increased in an Actinomycetes bacteria by transforming an Actinomycete with a vector that encodes a pantothenate kinase (panK) gene, and growing the bacteria under conditions that allow overexpression of said panK gene and in a medium supplemented with pantothenic acid. These engineered Actinomycetes produce more polyketides than wild-type Actinomycetes grown without pantothenic acid.
The Actinomycete can be a Streptomyces, more specifically Streptomyces venezuelae or Streptomyces peucetius.
The addition of recombinant DNA encoding a sugar transferase, glycosyltransferase, sugar synthase, glycosylsynthase, and/or sugar dehydratase directs polyketides synthesis of different products. The DesVII, DesVIII, elloramycin glycosyltransferase (ElmGT), and dNDP-D-hexose synthase dNDPD-hexose-4,6-dehydratase are some examples of sugar modifying enzymes that may be used in the present invention
The panK gene is expressed from a chromosomal integration, phage, plasmid, or shuttle vector comprising the panK gene operably linked to a promoter. The panK gene encodes a protein that phosporylates panthenoic acid to phosphopantetheine, more specifically the PanK protein is selected from table 2, one example being the E. coli panK gene.
CoA concentration is increased 2 fold in cells with PanK than cells without the PanK gene.
As used herein “polyketide” is defined as a large class of diverse compounds that are characterized by more than two carbonyl groups connected by single intervening carbon atoms, and also include derivatives of same, such as glycosylated polyketides. Polyketides include various substances having antibiotic, anticancer, cholesterol-lowering, and/or immunosuppressive effects.
Some examples of polyketides include: amphotericin B; antimycin A; brefeldin A; candicidin; epothilones; erythromycin; azithromycin; clarithromycin; erythromycin estolate; erythromycin ethylsuccinate; roxithromycin; ivermectin; josamycin; ketolides; leucomycins; kitasamycin; spiramycin; lovastatin; lucensomycin; macrolides; maytansine; mepartricin; miocamycin; natamycin; nystatin; oleandomycin; troleandomycin; oligomycins; rutamycin; sirolimus; tacrolimus; tylosin; oleandomycin; deoxyoleandolide; narbonolide; narbomycin and pikromycin.
Chiral compounds can include diasteriomers, entantiomers and mixtures (R-trans, R-cis, S-trans, and S-cis) unless a single chiral compound is specifically identified as the desired product. Biological reactions can produce a variety of compounds, diasteriomers, entantiomers and mixtures (R-trans, R-cis, S-trans, and S-cis) unless the reaction product is specifically identified as a single compound.
“Deoxy sugars” include deoxyhexose, 2-deoxyhexose, 6-deoxyhexose, rhamnose, fucose, and deoxyribose that are precursors to many biomolecules.
The terms “operably associated” or “operably linked,” as used herein, refer to functionally coupled nucleic acid sequences.
“Reduced activity” or “inactivation” is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, and the like. By “null mutant” or “null mutation” what is meant is that activity is completely inactivated. In one example, the control plasmid is inserted without the gene of interest. In another example the gene of interest is completely removed by recombination. Additionally, the gene of interest may be removed by inactivation, mutation, or truncation which eliminates activity.
“Overexpression” or “overexpressed” is defined herein to be at least 150% of protein activity as compared with an appropriate control species. Preferably, the activity is increased 100-500%. Overexpression is achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of a gene to the cell, up-regulating an existing gene, adding an exogenous gene, and the like.
The terms “disruption” and “disruption strains,” as used herein, refer to cell strains in which the native gene or promoter is mutated, deleted, interrupted, or down regulated in such a way as to decrease the activity of the gene. A gene is completely (100%) reduced by knockout or removal of the entire genomic DNA sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein.
The term “exogenous” indicates that the protein or nucleic acid is introduced from outside the organism or system, without regard to species of origin. For example, an exogenous peptide may be applied to the cell culture, an exogenous RNA may be expressed from a recombinant DNA transfected into a cell, or a native gene may be under the control of exogenous regulatory sequences or duplicated to increase expression.
As used herein “recombinant” is relating to, derived from, or containing genetically engineered material. Recombinant DNA can be carried on a vector or integrated into the chromosome of the host bacteria. Many vectors are known which can be used in a variety of species. Stable chromosomal integration methods are also well documented. In one method, recombinant DNA is integrated using P1 phage transduction and one-step inactivation based on λ red recombinase (Datsenko and Wanner, 2000). Briefly, a gene of interest is cloned adjacent to a selectable marker, both of which are flanked by chromosomal DNA sequence. When the recombinant DNA is inserted into a cell, the recombinant DNA integrates into the chromosome. Stably integrated DNAs are selected by serial plating on the selectable marker. Often the selectable marker is removed by recombination leaving the stable recombinant DNA in the host cell.
A gene or cDNA may be “optimized” for expression in E. coli, or other bacterial species using the codon bias for the species. Various nucleotides can encode a single peptide sequence. Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides which encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species.
“Pantothenate kinase” or “PanK” can be the E. coli panK gene or PanK protein sequence, or the panK gene can be from other species either wild-type or optimized for expression in Actinomycetes. There are over 650 PanK proteins available in the GenBank™ database. Non-limiting examples of PanK proteins are listed in Table 2.
E. coli K12 PanK
Shigella dysenteriae Sd197 PanK
Shigella boydii Sb227 PanK
E. coli O157:H7 PanK
Shigella flexneri 2a str. 301 PanK
Shigella flexneri 2a str. 2457T PanK
Salmonella typhimurium LT2 PanK
Salmonella enterica PanK
E. coli 53638 PanK
Yersinia pestis PanK
Yersinia intermedia ATCC 29909 PanK
Yersinia frederiksenii ATCC 33641 PanK
Erwinia carotovora PanK
Photorhabdus luminescens PanK
Haemophilus ducreyi PanK
Vibrio cholerae PanK
Mannheimia succiniciproducens PanK
Actinobacillus succinogenes PanK
Myxococcus xanthus PanK
E. coli B7A PanK
Mycobacterium PanK
Agrobacterium tumefaciens PanK
Bacillus subtilis PanK
E. coli B171 PanK
Streptococcus thermophilus PanK
Rattus norvegicus PanK1
Rattus norvegicus PanK2
Rattus norvegicus PanK3
Mus musculus PanK1 β
Mus musculus PanK2
Homo sapiens PanK1.α
Homo sapiens PanK1.β
Homo sapiens PanK1.γ
Homo sapiens PanK2.1 preproprotein
Homo sapiens PanK2.2
Homo sapiens PanK3
Homo sapiens PanK4
In calculating “% identity” the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity=number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova T A & Madden T L (1999) FEMS Microbiol. Lett. 174:247-250. The default parameters were used, except the filters were turned OFF. As of Jan. 1, 2001 the default parameters were as follows: BLASTN or BLASTP as appropriate; Matrix=none for BLASTN, BLOSUM62 for BLASTP; G Cost to open gap default=5 for nucleotides, 11 for proteins; E Cost to extend gap [Integer] default=2 for nucleotides, 1 for proteins; q Penalty for nucleotide mismatch [Integer] default=−3; r reward for nucleotide match [Integer] default=1; e expect value [Real] default=10; W word size [Integer] default=11 for nucleotides, 3 for proteins; y Dropoff (X) for blast extensions in bits (default if zero) default=20 for blastn, 7 for other programs; X dropoff value for gapped alignment (in bits) 30 for blastn, 15 for other programs; Z final X dropoff value for gapped alignment (in bits) 50 for blastn, 25 for other programs.
This program is available online at NCBI™ (ncbi.nlm.nih.gov/BLAST/).
Common restriction enzymes and restriction sites are found at NEB® (New England Biolabs®, neb.com) and Invitrogen® (invitrogen.com). ATCC®, American Type Culture Collection™ (atcc.org), DSMZ®, Deutsche Sammlung von Mikroorganismen and Zellkulturen™ (dsmz.de). KBIF®, Korean Biological Resource Center™ (kbif.kribb.re.kr), and WDCM®, World Data Centre for Microorganisms™ (wdcm.nig.acjp) have extensive collections of cell strains that are publicly available. NEB®, Invitrogen®, ATCC®, DSMZ®, KBIF®, and WDCM® databases are incorporated herein by reference.
Plasmids and strains used in certain embodiments of the invention are set forth in Table 3.
E. coli-Streptomyces shuttle vector
Streptomyces
venezuelae
Streptomyces venezuelae
Streptomyces venezuelae or S. venezuelae was originally isolated in 1948. Bacteria with similar genetic backgrounds are available through a variety of sources including ATCC® 10712, ATCC® 25508, CBS 650.69, DSM 41109, DSMZ® 40230, DSMZ® 40612, DSMZ® 40634, DSMZ® 40727, DSMZ® 41110, DSMZ® 41111, IFO 12595, IFO 13096, IMET 41356, IMRU 3534, IMRU 3625, ISP 5230, JCM 4526, KCC S-0526, NBRC 12595, NBRC 13096, NRRL B-2277, RIA 1288, VKM Ac-589, and other S. venezuelae strains available through different sources. All of these strains are expected to function in the experiments described given the similar genetic background.
“Shuttle vector” is a vector with two or more origins of replication for different species. This allows the vector to replicate in more than one species. The pIBR25 vector has an E. coli and S. venezuelae origin of replication. The E. coli origin allows high levels of plasmid replication in E. coli for cloning and genetic manipulation. The S. venezuelae origin allows expression of proteins in Streptomyces. Over 200 shuttle vectors are available through ATCC® alone. A shuttle vector can be generated for any species by cloning an origin of replication into a plasmid that already contains an origin for another species.
When plasmids are used, the effect of host/plasmid interaction is minimized by comparing three different systems consisting of: the host only, a plasmid expressing biologically active enzyme, and a control system with the expression vector alone.
E. coli was grown in Luria-Bertani broth (LB) with pH of 7.5 containing kanamycin and ampicillin. Cells were innoculated at 5×105 cells/ml and grown in a shaker flask, with rotation at 150 rpm, for 12 hours.
S. venezuelae cells were cultured in SPA medium containing per liter 1 gram of yeast extract, 1 gram of beef extract, 2 gram of tryptose, 10 gram of glucose, trace amount of ferrous sulfate, and appropriate antibiotics (25 μg/mL of thiostrepton or 50 μg/mL of kanamycin). Cells were innoculated at 5×105 cells/ml and grown in a shaker flask, with rotation at 150 rpm, for sampling. CoA and acetyl-CoA were assayed by HPLC according to the method described in Metabolic Engineering 6:133-139(2004). Metabolites were assayed by LC-MS system equipped with a reverse phase column (XTerra MS C18, Waters Co., MA) with the electrospray source with photodiode array detector. 10 mM ammonium acetate buffer solution was used as the mobile phase at a flow rate of 0.2 ml/min. The column was operated at 30° C.
S. venezuelae cells (wild type) were transformed by protoplast fusion using the E. coli-Streptomyces shuttle vector pIBR25 containing the panK gene from E. coli (pIBR25-PanK), added to the vector at the BamHI restriction sites. The transformed Streptomyces cells were unstable until the cloned gene was integrated into the chromosome. To select the stable transformants, several generations of transfer to the fresh media with selection pressure was performed. The transformed cells were then grown in pantothenic acid supplemeted medium (1 mg/ml), and CoA and acetyl-CoA levels were measured.
The data in
Similarly,
We then studied the separate effects of PanK overexpression from the effects of pantothenic acid supplementation by employing the appropriate controls.
Improving CoA and/or acetyl-CoA levels is a good first step, but it is also necessary to translate these gains into increased production of the desired secondary metabolite. To demonstrate that this goal was achievable, we stimulated deoxyoleandolide production in S. venezuelae. Cells were transformed with pIBR25-PanK or pIBR25. Stable transformants were selected by serial culture, as previously described.
One of the polyketides in the oleandomycin biosynthesis pathway (see
As an additional demonstration of increased production of secondary metabolites that rely on CoA or A-CoA, we studied the effect of increased intracellular CoA on narbonolide, as shown in
The polyketide biosynthesis pathway starting from coenzyme A (CoA) is shown in
This concept can be applied to the all the polyketides whose synthesis begins with CoA precursors. More examples of CoA compounds involving in polyketide biosynthesis are illustrated in the polyketides biosynthesis pathways in
Work is underway to expand the concept described herein to increase the production of various polyketides as illustrated in
All references are listed herein for the convenience of the reader. Each is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2006/037107 | 9/22/2006 | WO | 00 | 3/18/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/039177 | 4/3/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4766071 | Simon et al. | Aug 1988 | A |
| 20040199941 | San et al. | Oct 2004 | A1 |
| 20050089973 | Yocum et al. | Apr 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| PCTUS2006037107 | Apr 2008 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100047885 A1 | Feb 2010 | US |
