Patent Application
20090053769
The present invention relates to an E. coli-based total biosynthesis of a bioactive form of heterologous complex Nonribosomal Peptides (NRPs) from simple carbon and nitrogen sources.
Nonribosomal peptides (NRPs) encompassing vancomycin, cyclosporine A, echinomycin, and triostin A are celebrated components of a variety of microbial secondary metabolites possessing biological activities such as antibiotics, immunosuppressants, and antitumor agents immensely important for clinical use (1-4). This category's broad spectrum of natural products and its structural complexity are a result of them being biosynthesized by NRP synthetases (NRPSs) encoded in a single modulated megaenzyme ranging in size from 120 to 180 kDa. Each NRPS consists of three essential functioning domains: condensation, adenylation, and thiolation. These domains boast the ability to catalyze an amide bond formation using amino acids as building units for their peptide architecture (5-7). A single module of this megasynthetase may also carry a methylation domain for N-methylation of the peptide backbone and/or epimerization domain for switching an amino acid's stereochemistry during the peptide elongation process. The structural complexity and diversity of NRPs are largely attributed to these modules and methods in which they are synthesized.
Nature has provided a generous assortment of NRPs. However, the artillery of natural products as potential drugs or seeds of promising medication have their limitations due to insufficient material for investigative clinical trial. This obstacle can be attributed to the original host's low productivity or unavoidably expensive cost of multistep chemical syntheses to avoid undesirable byproducts and impart a favorable pharmacokinetic profile. During the past decade, many notable NRPs have been isolated from streptomycetes. Stimulated interests surrounding their mode of production compelled researchers to identify and sequence genes that are likely involved in this process. To understand the biosynthetic mechanisms, with aims for increasing the yield of production and intention of pursuing desired analogues, two agreeable expression systems using Streptomyces lividans and S. coelicolor as hosts with amplifiable expression vectors were developed by Hopwood et al. (8). These two systems have presented substantial results to provide lucid biological understanding of streptomycetes' proteins and the production of secondary metabolites, more markedly, polyketides (PKs), a category of natural products similar to NRPs in terms of how they are biosynthesized by modular macroenzymes (5). Although the contributions by the model hosts are influential to this field of research, construction of plasmid vectors and the cultivation of these cells are both time-consuming and problematic.
In recent years, progress has been made using E. coli as a surrogate host for gene expression of NRPSs and PK synthases (PKSs) (9). Positive results for heterologous production of anticipated compounds in E. coli were observed despite the use of Khosla's innovative but elaborate multiple-plasmid expression system (10-12). There are three great advantages of using E. coli as a heterologous host: (i) availability of a wealth of well-established molecular biological techniques for its genetic and metabolic manipulation, (ii) robust tolerance toward exogenous proteins and fast life cycle, and (iii) large-scale protein production ability, which will facilitate investigations for detailed reaction mechanisms of the biosynthetic pathway. Together, these advantages set the stage for engineering biosynthesis of desired compounds and its useful analogues through modification or mutation of the biosynthetic genes in a much shorter time frame.
The inventors recently established a de novo system by which echinomycin (1) and triostin A (2) (
The inventors estimated the production of compound 2 using small-scale cultivation and a liquid chromatography-mass spectrometer (LC-MS) for quantitative analysis of the product under select culture conditions. They also compared the de novo production while furnishing QXC to corroborate this chromophore's role as the priming unit for biosynthesis of the quinomycin antibiotics (18, 19). This simple and speedy approach provided for valuable information for maximizing the production titer.
The present invention provides examples of the de novo total biosynthesis of biologically active forms of heterologous NRPs in Escherichia coli (E. coli). The system can serve not only as an effective and flexible platform for large-scale preparation of natural products from simple carbon and nitrogen sources, but also as a general tool for detailed characterizations and rapid engineering of biosynthetic pathways for microbial syntheses of novel compounds and their analogs.
Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. Triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de novo production of triostin A by means of high cell density fed-batch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with small-scale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy approach provided for valuable information for maximizing the production titer. The entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.
The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.
An artillery of natural products as potential drugs or seeds of promising medication have their limitations due to insufficient material for investigative clinical trial. Many soil and marine bacteria are not amenable to cultivation and require time-consuming, highly optimized conditions for mass-production of desired secondary metabolites for clinical and commercial use (22). Therefore, a fast, simple system for heterologous production of natural products is desired.
The present invention demonstrates what the inventors believe to be the first example of the de novo total biosynthesis of biologically active forms of heterologous NRPs in Escherichia coli (E. coli). The system can serve not only as an effective and flexible platform for large-scale preparation of natural products from simple carbon and nitrogen sources, but also as a general tool for detailed characterizations and rapid engineering of biosynthetic pathways for microbial syntheses of novel compounds and their analogs.
Recently, the development of E. coli as a vehicle for heterologous metabolite biosynthesis has seen considerable progress. Perhaps, the greatest advantage of using E. coli is the wealth of knowledge available on its metabolic pathways and genetic make-up, as well as the availability of well-established techniques for its genetic manipulation. Additionally, the ease of E. coli fermentation makes this organism particularly suitable for metabolite overproduction. Its tolerance toward heterologous protein productions and short doubling time are also vital to the efforts. However, the inventors believe no biologically active complex natural product synthesized by heterologous polyketide synthase (PKS), NRP synthetase (NRPS) or mixed PKS/NRPS has been obtained de novo from E. coli. Therefore, the inventors established an E. coli system capable of total biosynthesis of biologically active forms of NRPs.
Compound 1 is an NRP isolated from various bacteria, including Streptomyces lasaliensis (23), that belongs to the large family of quinoxaline antibiotics that have quinoxaline chromophores attached to the C2-symmetric cyclic depsipeptide core structure. Great interest in this group of compounds stems from its potent antibacterial, anticancer and antiviral activities. Many, including 1 and triostin A 2, exhibit nanomolar potency (24). Also, 1 contains unique chemical structures, including the quinoxaline-2-carboxylic acid (QC) moiety and the thioacetal bridge, whose biosynthetic mechanisms remain unknown.
The inventors isolated the echinomycin biosynthetic gene cluster from the S. lasaliensis linear plasmid in accordance with Watanabe, et al. 2006. DNA sequence analysis of the 36 kilobase-long cluster revealed the presence of eight genes that appeared responsible for the QC biosynthesis (ecm2-4, 8, 11-14), five genes for the peptide backbone formation and modifications (ecm 1, 6, 7, 17, 18) and a resistance gene (ecm16) (
Curiously, the aryl carrier protein (ArCP) required for incorporating QC into 1 was absent from the cluster. However, as in the triostin A biosynthesis (28), the inventors expected the adenylation (A) domain-containing Ecm1 to activate and transfer QC to the phosphopantetheine arm of FabC, the fatty acid biosynthesis acyl carrier protein (ACP). The first module of the bimodular NRPS Ecm6 can accept QC-S-FabC as the starter unit, while Ecm6 and the second NRPS Ecm7 catalyze seventeen chemical reactions for the peptide core formation (Scheme 1b). Ecm7 contains a terminal TE domain that appears to homodimerize and cyclorelease the peptide chain (Scheme 1c)(29). The cyclized product can then become the substrate for an oxidoreductase Ecm17 that can catalyze an oxidation reaction within the reducing cytoplasmic environment to generate the disulfide bond in 2.
The last step of echinomycin biosynthesis involves an unusual transformation of the disulfide bridge of 2 into a thioacetal bridge (30). Ecm18, which is highly homologous to a known S-adenosyl-L-methionine (SAM)-dependent methyltransferase, is thought to be responsible (
For the E. coli production of 1, after confirming the feasibility of expressing each of the fifteen S. lasaliensis genes (ecm 1-4,6-8, 11-14, 16-18, fabC) in E. coli, the inventors assembled the fifteen genes along with the Bacillus subtilis phosphopantetheine transferase gene sfp, known to efficiently phosphopantetheinylate heterologous ACPs and T domains (36), into three separate plasmids with each gene carrying its own T7 promoter, ribosome binding site and T7 transcriptional terminator (13). The inventors chose this multi-monocistronic arrangement for the multi-gene assembly, not only for simplifying the assembly process but also for minimizing previously observed premature terminations and mRNA degradation in transcribing excessively long polycistronic gene assemblies (37). Moreover, orthogonal origins of replication and antibiotic resistance genes were used to ensure the stable retention of all three plasmids in E. coli (38). The E. coli strain BL21 (DE3) transformed with the three plasmids was subjected to eight-day long fed-batch fermentation in minimal medium. When the culture extract was fractionated by high-performance liquid chromatography (HPLC) and analyzed by electrospray ionization-mass spectrometry (ESI-MS) (
When introducing any exogenous biosynthetic pathway into E. coli, the toxicity of the biosynthetic product can impair the host. This problem can be circumvented by introducing a self-resistance mechanism into E. coli that confer resistance without destroying the product. For echinomycin biosynthesis, the homology between Ecm16 and daunorubicin resistance-conferring factor DrrC (39), and the similarity of the mode of action between 1 and daunorubicin (40) suggested that Ecm16 achieves non-destructive resistance against 1 in S. lasaliensis. Subsequently, the inventors were able to demonstrate that ecm16 conferred echinomycin resistance to BL21 (DE3). Also, when ecm16 was absent from the system, the growth of the host was hampered, suggesting that sufficient amounts of 1 and 2 would have been unattainable without the self-resistance mechanism in place.
The following examples are intended to illustrate, but not to limit, the scope of the invention. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.
Chemicals. Antibiotics were used at the following concentrations: carbenicillin 100 μg/mL, kanamycin 50 μg/mL, and spectinomycin 50 μg/mL. QXC was purchased from Sigma-Aldrich. Other chemical reagents were purchased at the highest commercial quality and used without further purification.
Bacteria Strains and Media. Common procedures, including plasmid manipulation, transformation, and other standard molecular biological techniques, were carried out as previously described in Sambrook and Russell (41). E. coli DH5 cc purchased from Invitrogen was used for plasmid amplification and grown in Luria-Bertani (LB) medium or M9 minimal medium (41) at 37° C. Overproduction of recombinant proteins for production of compound 2 was carried out in E. coli BL21 (DE3) (Invitrogen). Cell growth was monitored using optical density and measured at 600 nm (OD600), and isopropyl 1-thio-β-D-galactopyranoside (IPTG) was used to induce. Feed media used for fermentation contained 430 g/L of glucose, 3.90 g/L of MgSO4, 10 g/L of alanine, trace metal (0.278 g/L of FeCl3.6H2O, 0.130 g/L of ZnCl2, 0.013 g/L of CaCl2.2H2O, 0.021 g/L of NaMoO4.2H2O, 0.190 g/L of CuSO4.5H2O, 0.024 g/L of H3BO3), and vitamins (0.00420 g/L of riboflavin, 0.05456 g/L of pantothenic acid, 0.06078 g/L of nicotinic acid, 0.014140 g/L of pyridoxin, 0.00062 g/L of biotin, and 0.00048 g/L of folic acid).
Assembling the plasmid-based echinomycin biosynthetic gene cluster. Initially, each ORF was cloned individually into pET28b (Novagen)-derived pKW409 as either a Nde I-EcoR I or a Nde I-Xho I fragment prepared by PCR (13). In pKW409, the single Xba I recognition site was moved to the 5′ side of the T7 promoter, and a Spe I recognition site was created at the 3′ side of the T7 terminator. The assembly formation exploited the compatibility of the cohesive ends generated by Xba I and Spe I digestion. The cassette arrangement not only facilitated evaluation of the expression level of each gene individually, but also was necessary for rapid construction of the multi-monocistronic gene assemblies (13).
Thioacetal formation assay. The assay mixture containing 10 μM Ecm 18 and 10 mM SAM in 0.1 M Tris-HCl pH 7.2 was pre-incubated at 30° C. for 5 min. After addition of 2 to the final concentration of 1 mM, the reaction was run at 30° C. for five minutes before being terminated by addition of 10% (w/v) SDS. The reaction mixture was extracted with ethyl acetate, and the extract was concentrated in vacuo to give a white residue. Compound 1 and 2 were isolated from the extracts using PTLC (5% MeOH/CHCl3). The isolated samples of 1 and 2, along with the mixture were analyzed independently by HPLC-MS Alltech 2.1×100 mm C18 reverse-phase column. Samples were subjected to a linear gradient of 5 to 95% CH3CN (v/v) in H2O supplemented with 0.05% (v/v) formic acid over 60 min at a flow rate of 0.15 ml/min at room temperature. Compound 1 eluted at 41.65 min under the condition described above, and gave characteristic ions at m/z=1139.24 [M+K]+, 1123.34 [M+Na]+, 1101.29 [M+H]+ and 1053.38 [M-SCH3]+. Similarly, 2 eluted at 41.15 min and gave characteristic ions at m/z=1125.30 [+K]+, 1109.44 [M+Na]+ and 1087.49 [M+H]+. These results matched well with the analyses of the authentic samples of 1 and 2 (
E. coli production of 1 and 2. BL21 (DE3) transformed with pKW532/pKW538/pKW541 and pKW532/pKW539/pKW541 for the production of 1 and 2, respectively, was incubated at 37° C. overnight in 2 ml Luria-Bertani (LB) medium and subsequently in 100 ml M9 minimal medium. The entire culture was used to inoculate 1.5 liters of M9 minimal medium kept at 37° C., pH 7.0 by the BioFlo110 fermentor system (New Brunswick Scientific). Once the glucose was exhausted from the medium as indicated by a sudden increase of the dissolved oxygen level, feeding of the feed media (42) was initiated. When the culture reached an OD600 of 11, the temperature was reduced to 15° C., and isopropylthio-β-D-galactoside (IPTG) was added to the final concentration of 200 μM. After eight to twelve days of incubation, the culture was centrifuged to separate the supernatant and the cells. The supernatant and the cell pellet were extracted with ethyl acetate and acetone, respectively. The extracts were combined and concentrated in vacuo to give an oily residue, which was fractionated by silica gel flash column chromatography with 50% MeOH/CHCl3. The fractions containing the target compound were collected and further purified by a series of preparative thin-layer chromatography (PTLC: i. 50% EtOAc/hexane; ii. 2-butanone; iii. 5% MeOH/CHCl3) to afford purified samples 1(43) and 2(44). The isolated compounds were fully characterized by HPLC-MS, MS/MS, 1H NMR and 1H NMR TOCSY.
Echinomycin resistance assay. E. coli echinomycin resistance was determined using BL21 (DE3) transformed with pKW409 carrying ecm 16. The transformant was incubated in 3 ml LB medium at 37° C. for 5 hours. The culture was spread on LB agar plates containing two different concentrations of echinomycin (10 and 100 μg/ml) supplemented either with or without 300 μM IPTG. The plates were incubated at 37+C overnight to determine colony formation. Accession codes. The echinomycin biosynthetic gene cluster sequence has been deposited in DNA Data Bank of Japan with the accession numbers AB211309 and AB211310.
For de novo Production of Compound 2 in E. coli Using Shake Flask. Three plasmids, pKW532 (ecm2-4, 8, and 11-14), pKW539 (ecm1, 16, 17, fabC, and sfp) and pKW541 (ecm6 and 7) were transformed into E. coli BL21 (DE3) (13). The cells were incubated at 37° C. overnight in 2 mL of LB medium supplemented with carbenicillin, spectinomycin, and kanamycin. Subsequently, 0.5 mL of the culture was used to inoculate 50 mL of M9 minimal medium with the antibiotics described above. The culture was grown at 37° C. until its OD600 reached 0.3-0.6, at this point, the temperature was reduced to 15° C. and IPTG was added at a final concentration of 200 μM. Simultaneously, 10 mL of a feed medium (11) was added and continuously shaken at 150 rpm for 8 days.
For QXC Fed Production of Compound 2 in E. coli Using Shake Flask. Two transformants were prepared: a culture transformed with three plasmids pKW532/pKW539/pKW541 and another transformed with only two plasmids pKW539 and pKW541, the biosynthetic gene cluster for compound 2 (45, 46). The described constructs were transformed into BL21 (DE3) and cultivated for production of 2. Culture conditions for BL21 (DE3) transformed with pKW532/pKW539/pKW541 are the same as those used for de novo production. Two feed methods were explored for cells transformed with pKW532/pKW539/pKW541. A single dose of QXC was added to our culture at the point of gene expression for a final concentration of 0.1 mg/mL. Subsequent experiments where a daily dose of QXC at 5 mg per feed initiated at the point of gene expression were also examined. Production of 2 in LB medium under the same culture conditions with a daily supply at 5 mg of QXC per feed for cultures transformed with pKW532/pKW539/pKW541 was also explored. BL21 (DES) transformed with pKW539/pKW541 was incubated at 37° C. overnight in 2 mL of LB medium supplemented with spectinomycin and kanamycin. Subsequently, 0.5 mL of the culture was used to inoculate 50 mL of M9 minimal medium with added antibiotics as described above. All other culture conditions matched those described for de novo production of 2. A daily feed of QXC in the amount of 5 mg per feed was initiated at the induction point.
Quantitative Analysis of Compound 2 Production. Upon induction of gene expression, 1 mL of the culture was collected and centrifuged to pellet the cell and harvest the supernatant for analysis. The supernatant was extracted with ethyl acetate (2×1 mL) and concentrated in vacuo. The resultant residue was dissolved with 100 μL of methanol and 15 μL of the resulting mixture was analyzed by LC-MS using an Alltech 2.1×100 mm C18 reverse-phase column. Samples were separated on a linear gradient of 5% to 95% CH3CN (v/v) in H2O supplemented with 0.05% (v/v) formic acid over 40 min at room temperature and a flow rate of 0.1 mL/min. To quantitate the production of 2, a Finnigan LCQ Deca XP mass spectrometer equipped with an electrospray probe operating on positive mode was used. We prepared a standard curve with a range between 1 ng and 10 μg of reference compound 2. Mass spectra collected in single ion monitoring mode were obtained by monitoring two ions ([M+H]+ and [M+Na]+, m/z 1087 and 1109, respectively) observed at retention time 17.5 min of bp070298yt00001 the LC. The following optimized values were employed for data acquisition: capillary temperature 275° C.; spray voltage 5 kV; source current 80 μA; capillary voltage 12 V; sheath gas N2 flow 60 (arbitrary units). Accumulated compound 2 in culture was then extrapolated using our standard curve following the steps described above.
In our previous report, E. coli expression of a multiple-plasmid system in M9 minimal medium containing the complete biosynthetic pathway of 2 produced 0.6 mg/L of isolated compound via fed-batch fermentation. Although de novo production of NRP, compound 2, was a successful achievement, its titer was modest in amount despite the use of the fed-batch fermentation process (Table 1). To address the low productivity by means of a simple and quick procedure, we analyzed the production of 2 using small-scale shake flask culture (
aCulture vessels are described in Materials and Methods.
bDaily feed of QXC was supplied at 5 mg/day post induction until harvest of culture. A single dose of QXC was supplied once at the same amount during the point of induction. QXC was not supplied for de novo production of compound 2.
cThe reported level of compound 2 is shown in milligrams of product per liter of culture.
dIsolated quantity of 2 from fed-batch fermentation.13
These findings suggest an indispensable interaction between QXC's biosynthetic protein or proteins with either ecm1 or ecm1 and fab C. Improving modest yields from de novo production of 2 by simply adding commercial QXC has confirmed its assembly as the bottleneck. These findings have provided further evidence and narrowed QXC's role as the priming unit for biosytnthesis of 2+Facile expression of the intact biosynthetic gene cluster for compound 2 in a heterologous host, E. coli has eased our attempts at garnering data to provide a more comprehensive picture of 2's dependence on QXC. Although we have identified and presented the stereochemical assignment for the β-hydroxytryptophan intermediate in the QXC biosynthetic pathway (19), it will require additional investigation in order to aid and circumvent the inherent challenges of biological studies and metabolic engineering for de novo production of 2 in E. coli to ultimately increase its yield. Further elucidation of the pathway is ongoing.
A pH and cell growth profile is shown in
Mounting evidence has revealed how fed-batch fermentation technology for metabolic engineering may possess the capability of producing a more substantial level of 2 by merely supplementing exogenous starting unit during biosynthesis of compound 2. Equally, testing of precursor-directed biosynthesis by means of feeding an assortment of chromophore to the heterologous expression system may provide us with a combinatorialy engineered biosynthesis of unnatural quinomycin antibiotics. This tolerance is indispensable to the biosynthetic research field because of its simplicity and speedy assembly line.
In conclusion, the present invention demonstrates, for what the inventors believe to be the first time, the viability of E. coli-based total biosynthesis of a bioactive form of heterologous complex NRPs from simple carbon and nitrogen sources, paving the way to developing an economical, general platform for one-pot mass-production of natural products and their analogs. The system shows that using a multi-plasmid, multi-monocistronic gene assembly is a straightforward, highly stable and easily modifiable approach for establishing and engineering exogenous biosynthetic pathways in E. coli. With the use of appropriate orthogonal selection markers and origins of replication, in combination with other potential approaches, such as chromosome integration (45), introducing even larger, more complex biosynthetic pathways seems attainable. Combining these current efforts with the successes in introducing other PKS(46,48) and mixed PKS-NRPS(42) pathways into E. coli and engineering of PKS(49) and NRPS(50) should help broaden the scope of E. coli-based heterologous mass-production of a wide range of natural products and their analogs.
The present invention also substantiated QXC as the primer unit of 2 by using a heterologous host for its biosynthesis while supplying the culture with the chromophore. Under optimal conditions, the cultures were then subsidized with QXC to afford 13 mg/L of compound 2, an increase of more than 130-fold relative to its de novo production in shake flask fermentation. By using LC-MS to analyze small-scale culture, the inventors obtained indispensable information in a much shorter time frame. The disclosed method has considerable importance for potential endeavors to creating a quinomycin antibiotic library.
Obviously, many modifications and variation of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following references are cited herein. The entire disclosure of each reference is relied upon and incorporated by reference herein.
The present application claims the benefit of the filing date of U.S. Provisional Application No. 60/944,620, filed Jun. 18, 2007, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with support by grants from NIH GM 075857-01, American Cancer Society grant RSG-06-010-01-CDD, University wide AIDS Research Program ID05-USC-055, and Grant-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (A) 17208010.
| Number | Date | Country | |
|---|---|---|---|
| 60944620 | Jun 2007 | US |
