Patent Application
20160304884
In modern biotechnology, the fermentation of genetically modified microbes has been industrialized for the production of large biomolecules such as protein or DNA. In these instances, only a limited number of genetic modifications is introduced into the cells. With the recent developments in genomics, microbes are now used to produce valuable chemical compounds by metabolic engineering and to generate novel biological phenotypes by synthetic biology. These processes often involve the modifications of one or more intricate biological pathways which necessitate the manipulations of multiple genes.
In bacteria, plasmids are routinely used in metabolic engineering and synthetic biology simply because these are easily manipulated and can have multiple copies in cells. There are however, significant limitations restricting the utility of multiple plasmids in large scale fermentation processes. Firstly, the co-existence of two or more incompatible plasmids will invariably be regulated resulting in unstable copy numbers whereby some of these plasmids will be lost over prolong periods of fermentation. One approach to circumvent this is to use plasmids that are naturally compatible. However, there are only limited numbers of these plasmids with distinct copy numbers. These plasmids have different replication mechanisms which are known to respond differently to environmental cues making the simultaneous use of these plasmids a challenge. Secondly, because they are non-essential for growth, plasmids may be lost during the scaling-up process where cells are known to be metabolically stressed by the over-expression of genes carried by the plasmid. One approach to circumvent this is to use antibiotics to exert selection pressures. Unfortunately, the use of antibiotics is both costs prohibitive, unstable in long term growth and faces significant regulatory issues.
Hence, to be industrially relevant, combinatorial panels of plasmids which can stably co-exist in a cell and grow under selection pressure without the use of antibiotics are needed.
Described herein is the development of a multi-plasmid system (Compatible Antibiotic-free Multi-Plasmid ystem, CAMPS) for the expression of one or more (multiple; a plurality) nucleic acid sequences of interest (e.g., genes of interest (GOI)) carried by a one or more (multiple; a plurality) multi-compatible plasmids in specially engineered host cells and grown in antibiotic-free medium.
Accordingly, in one aspect the invention is directed to a method of expressing a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc.) of nucleic acid sequences comprising maintaining a (one or more) host cell comprising one or more, and preferably, two or more plasmids. In a particular aspect, the two or more plasmids are compatible. The host cell lacks one or more conditional essential genes, and the two or more plasmids comprise the two or more nucleic acid sequences to be expressed and the sequences of the one or more conditional essential genes, and each plasmid further comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the Ori of each plasmid. The host cell is maintained under conditions in which the two or more nucleic acid sequences and the one or more conditional essential genes are expressed in the host cell, thereby expressing the two or more nucleic acid sequences.
In another aspect, the invention is directed to a host cell comprising two or more plasmids, wherein each plasmid comprises one or more conditional essential genes (CEGs). In another aspect, the invention is directed to a host cell wherein (i) the host cell lacks one or more conditional essential genes, and (ii) the two or more plasmids comprise the one or more conditional essential genes. The plasmids can further comprise an Ori that is identical except for one or more loop sequences in the Ori of each plasmid.
In yet another aspect, the invention is directed to a plurality of plasmids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc), such as a panel of plasmids. In one aspect, the plurality of plasmids comprises two or more plasmids.
In another system, the invention is directed to a system for expressing two or more nucleic acid sequences comprising a host cell and two or more plasmids, wherein the host cell lacks one or more conditional essential genes and the two or more plasmids comprise the one or more conditional essential genes, and each plasmid comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the Ori of each plasmid.
In yet another embodiment, the invention is directed to a method of preparing a library of compatible plasmids comprising introducing into a host cell at least two plasmids wherein each plasmid comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the Ori of each plasmid; and maintaining the host cell under conditions in which the plasmids are replicated in the host cell, thereby by preparing a library of compatible plasmids.
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
A description of example embodiments of the invention follows.
Described herein is the development of a multi-plasmid system (Compatible Antibiotic-free Multi-Plasmid System, CAMPS) for the expression of one or more (multiple; a plurality) nucleic acid sequences of interest (e.g., genes of interest (GOI)) carried by one or more (multiple; a plurality) multi-compatible plasmids in specially engineered host cells and grown in antibiotic-free medium. This was designed based on modifying a parental plasmid where the recognition sites controlling plasmid copy number were specifically engineered to produce a library of compatible plasmids. Thus, these plasmids share the same replication mechanism but vary in copy numbers. In order to maintain these multiple plasmids without using antibiotics, multiple conditional essential genes (CEG) from the host genome were grafted into these plasmids. As a result, all of these co-existing plasmids carrying the CEG were maintained in a host where the corresponding CEGs were knocked out from the host's genome during fermentation. The combination of multiple engineered compatible plasmids using the same replication mechanism co-existing in an antibiotic free fermentation condition has broad utility in metabolic engineering and synthetic biology.
Accordingly, in one aspect the invention is directed to a method of expressing a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc.) of nucleic acid sequences comprising maintaining a (one or more) host cell comprising one or more, and preferably, two or more plasmids. In a particular aspect, the two or more plasmids are compatible. As used herein, compatible plasmids are plasmids that can stably co-exist in a host (e.g., host cell) and/or can grow under selection pressure. In a particular aspect, compatible plasmids can further grow without the use of antibiotics. The host cell lacks one or more conditional essential genes, and the two or more plasmids comprise the two or more nucleic acid sequences to be expressed and the sequences of the one or more conditional essential genes, and each plasmid further comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the Ori of each plasmid. The host cell is maintained under conditions in which the two or more nucleic acid sequences and the one or more conditional essential genes are expressed in the host cell, thereby expressing the two or more nucleic acid sequences.
In another aspect, the invention is directed to a method of expressing two or more nucleic acid sequences comprising introducing into a host cell two or more plasmids, wherein (i) the host cell lacks one or more conditional essential genes and (ii) the two or more plasmids comprise the two or more nucleic acid sequences and the one or more conditional essential genes, and each plasmid comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the Ori of each plasmid. The host cell is maintained under conditions in which the two or more nucleic acid sequences and the one or more conditional essential genes are expressed in the host cell, thereby expressing the two or more nucleic acid sequences.
In another aspect, the invention is directed to a host cell comprising two or more plasmids, wherein each plasmid comprises an origin of replication that is identical except for one or more loop sequences in the Ori of each plasmid. In some aspects, the host cell lacks one or more conditional essential genes. In other aspects, the plasmids can further comprise one or more conditional essential genes. In yet another aspect, the plasmids can comprise one or more conditional genes that are lacking in a (one or more) host cell.
In another aspect, the invention is directed to a host cell comprising two or more plasmids, wherein each plasmid comprises one or more CEGs. In another aspect, the invention is directed to a host cell wherein (i) the host cell lacks one or more conditional essential genes, and (ii) the two or more plasmids comprise the one or more conditional essential genes. The plasmids can further comprise an ORi that is identical except for one or more loop sequences in the Ori of each plasmid.
In yet another aspect, the invention is directed to a plurality of plasmids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc), such as a panel of plasmids. In one aspect, the plurality of plasmids comprises two or more plasmids.
In another aspect, each plasmid comprises an Ori that is identical except for one or more loop sequences in the Ori of each plasmid. The one or more plasmids can further comprise one or more CEGs. In a particular aspect, the plasmid comprises one or more CEGs that a (one or more) host cell lacks.
In another aspect, each plasmid comprises one or more conditional essential genes of a host cell. In particular aspects, the one or more plasmids comprise the one or more CEGs that a (one or more) host cell lacks. The plasmids can further comprise an Ori that is identical except for one or more loop sequences in the Ori of each plasmid.
In another aspect, the invention is directed to a system for expressing two or more nucleic acid sequences comprising a host cell and two or more plasmids, wherein the host cell lacks one or more conditional essential genes and the two or more plasmids comprise the one or more conditional essential genes, and each plasmid comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the Ori of each plasmid.
In yet another embodiment, the invention is directed to a method of preparing a library of compatible plasmids comprising introducing into a host cell at least two plasmids wherein each plasmid comprises an origin of replication (Ori) that is identical except for one or more loop sequences in the On of each plasmid; and maintaining the host cell under conditions in which the plasmids are replicated in the host cell, thereby by preparing a library of compatible plasmids.
As described herein, the plasmids in the compositions and methods are compatible. As used herein, “compatible” plasmids refer to plasmids that when present in a host cell do not inhibit the replication of one another in the host cell. In addition or in the alternative, compatible plasmids are plasmids that can stably co-exist in a host (e.g., host cell) and/or can grow under selection pressure. In a particular aspect, compatible plasmids can further grow without the use of antibiotics. Each plasmid (e.g., in the host cell, the panel of plasmids, the system and/or the method) can further comprises an origin of replication (Ori), wherein the Ori of each plasmid is identical except for one or more nucleotides (bases) in the Ori sequence. In one aspect, the Ori of the two or more plasmids have a stem-loop structure wherein the two or more plasmids are identical except for one or more of the loop sequences in the Ori of each plasmid. The Ori of each plasmid can differ by one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) nucleotides. In a particular aspect, the Ori of each plasmid comprises 3 loops and one or more nucleotides in one or more of the loops (e.g., a first loop, a second loop and/or a third loop) of the On of each plasmid differs. In particular aspects, the Ori of each plasmid comprises 3 loops and the sequence of the second loop of the Ori of each plasmid differs (e.g., differs by one nucleotide; differs by two nucleotides, differs by three nucleotides, etc.). In one aspect, The method of any one of the preceding claims wherein at least one plasmid comprises a recognition site for RNase H (e.g., located near or next to the stem-loop structure of the Ori).
In addition, at least one plasmid in the host cell can further comprises one or more cloning sites, a nucleic acid sequence encoding a marker, and/or one or more nucleic acid sequences to be expressed in the host cell.
A variety of plasmids can be used in the compositions and methods provided herein. In particular aspect, the one or more plasmids comprise one or more of the following features; synthesize RNA primer for the initiation of replication (e.g., RNA II) with stem-loop structures (e.g., three); synthesize another antisense RNA for the regulation of replication (e.g., RNA I) with complementary sequences and stem-loop structures; synthesize initiator of replication (e.g., repZ protein) for the initiation of replication (e.g., RNA II) with stem-loop structures in its mRNA; synthesize another antisense RNA (e.g., Inc RNA) for the regulation of the translation of replication initiator (e.g., repZ protein) with complementary sequences and stem-loop structures; synthesize a polypeptide or RNA as the initiator for replication where the mRNA of the polypeptide or the RNA initiator has stem-loop structure(s); synthesize another antisense RNA with complementary sequences and stem-loop structure(s) to regulate the initiator; and/or comprise one or more loop sequences in an Ori that differ by one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) nucleotides. Specific examples of plasmids include ColEl, pl5A, Incla (e.g., Collb-P9), pMV158, Inc18 (e.g., pIP501, pSM19035) and the like.
Any of a variety of host cells can be used in the methods and compositions provided herein. In one aspect, the host cell is an auxotroph host cell. For example, the host cell can be a prokaryotic host cell (e.g., E. coli).
As used herein, a “conditional essential gene” (CEG) is a gene of a host cell that is needed for growth of the host cell under one or more particular growth conditions (e.g., growth in minimal media), such as a metabolic gene. The one or more conditional essential genes (CEGs) are essential when the host cell is cultured in a selection culture media but not in a non-selection culture medium. As used herein a “selection (e.g., minimal) culture (growth) media” is a culture medium that lacks one or more essential components (e.g., the minimal necessities) for growth of the cell (e.g., a host cell that lacks one or more conditional essential genes). As used herein a “non-selection (e.g., rich) media correct is a media that includes one or more essential components for the growth of a cell (e.g., a host cell which lacks one or more conditional essential genes). In one aspect, the selection culture media comprises one or more sugars. In another aspect, the one or more sugars are glucose, glycerol or a combination thereof.
In other aspects, the one or more conditional essential genes encode one or more polypeptides in one or more biochemical pathways of the host cell such as a metabolic pathway of the host cell. In particular aspects, the one or more conditional essential genes encode one or more metabolic enzymes of the host cell. Examples of metabolic enzymes include aroA, aroB, aroC, pdxH, pyrF, proC, argB, arC, argH or a combination thereof.
Examples of CEGs are listed below.
List of Conditional Essential Genes that are Essential in Glucose or Glycerol Minimal Medium:
As described herein the host cell can lack one or more CEGs. In particular aspects, the host cell lacks 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc, CEGs. Each conditional essential gene can be inserted into a separate plasmid.
The methods provided herein can further comprising adding one or more moieties e.g., chemicals, produced by one or more polypeptides or intermediates thereof to the culture media, wherein the one or more polypeptides are encoded by the one or more conditional essential genes. In particular aspects, the moieties are needed for the growth of a host cell (e.g., an auxotroph host cell, for example, that lacks CEGs—the lack of CEGs results in the lack of essential metabolites for growth). In particular aspects, the one or moieties can be (i) the direct product (e.g., metabolite) of one or more polypeptides encoded by one or more CEGs that are or that can be converted to the essential metabolites lacking in the host cell (e.g., the auxotroph host cell) or (ii) the other metabolites that are or that can be converted to the essential metabolites (e.g., downstream of CEG and upstream of the essential metabolites).
In a particular aspect, the one or more intermediates are downstream in a biochemical (e.g., metabolic) pathway of the one or more CEGs. In other aspects, the one or more chemicals comprise one or more metabolites produced by the one or more polypeptides or intermediates thereof. For example, the one or more products can comprise pyridoxal 5′-phosphate (PLP), proline (PRO), uridine monophosphate (UMP), arginine (ARG), shikimate (SK), ornithine (OR) or a combination thereof.
In some aspects, the moiety (e.g., chemical such as a metabolite) for a CEG or a set of CEGs from a sequential metabolic pathway can be the direct product or the metabolite downstream of the CEG or the set of CEGs. Examples of such chemicals are provided below.
In particular aspects of the methods provided herein, the two or more nucleic acid are expressed under antibiotic-free conditions. In other aspects, the one or more nucleic acid sequence is one or more genes. In yet other aspects, the one or more genes are overexpressed by the host cell.
A scalable compatible antibiotic-free multi-plasmid system (CAMPS) for metabolic engineering and synthetic biology
Results
Overview of CAMPS
To develop this scalable system (CAMPS) for metabolic engineering and synthetic biology, an integrated: 1) compatible plasmids and 2) conditional host cell system was designed and engineered specifically. These two components functions as one to achieve the desired outcome of an antibiotic free multi-plasmid expression system for the scale-up production of valued compounds by controlling the expressions of multiple genes (FIG. I).
Engineering of Compatible Plasmids
The replication and copy number of a class of plasmid (such as P15A and ColE1) is tightly controlled by the trans-acting factor RNA I as illustrated in
Described herein is the generation of a set of plasmids (
The step in replication, which can result in plasmid incompatibility, is the annealing of RNA I and RNA II to the recognition sites. As shown herein, altering the loop sequences made it possible to modify the recognition (
Precisely which nucleotide and the number of nucleotides on the loop to be modified was determined empirically as the effect of loop sequence on complex stability did not appear to vary simply with the number of potential Watson-Crick base-pairs that can be formed. For example, sequences that produce inverted loops can result in extremely low copy numbers. In addition, single nucleotide changes in the sequences of RNA I/RNA II overlap can alter the affinity of their interaction but can maintain the complementarity of the sequences. This built-in tolerance to point mutations is thought to facilitate the mutational fine-tuning of the system to maintain the existence of compatible plasmids and exclude incompatible plasmids with only minor changes. As a result, with single nucleotide changes, the incompatibility properties can only be altered to a limited extent. Thus, to modify the sequences to achieve compatibility of multiple plasmids experimentation was needed since it was not obvious by in silico prediction.
To create a large plasmid library whereby members are not only compatible with the parental plasmid but also with other members in the library, the engineered sites on the loops should be sufficiently different between different plasmids. Targeting at the loop sequences, a number of modifications to enable these plasmids to be compatible were empirically identified,
At the same time, because the synthesis of RNA I and RNA II of the same plasmid share the same DNA template, the copy number control mechanism can be retained for each member. Thus, by engineering the Ori sites, plasmids that shared the same replication mechanism as the parental which were similarly regulated upon environmental changes were generated.
To demonstrate the idea, the second loop in the stem-loop structures of the origin of replication of the vector pAC vector, p15A Ori was generated. A series of new plasmids with unique 2nd loop sequences were constructed by site-directed mutagenesis and the screened plasmids showed a range of copy numbers. Selected plasmids were then tested and confirmed to be compatibility with the original one and with each other.
Construction of Artificial Multi-Compatible Plasmids
In order to construct multiple compatible plasmids based on the designs described above, site-directed mutagenesis was carried out to mutate the 2nd loop of p15A Ori (
Compatibility Test of Multi-Compatible Plasmids
As a proof-of-concept, multiple plasmids with different engineered On sites were tested for compatibility. The original site (p15A) and two engineered sites (p15AL2-5 and p15AL2-8) were inserted into different plasmids together with various antibiotic resistant genes (
Firstly, a kanamycin (Kan) resistant plasmid with the original Ori site (T7-dxs-Kan-p15A) was co-transformed with each of the three Ori sites carrying the chloramphenicol (Cam) resistant plasmid (T7-ADS-ispA-Cam-p15A, T7-ADS-ispA-Cam-p15AL2-5 or T7-ADS-ispA-Cam-p15AL2-8). Both Kan and Cam were used initially to maintain the plasmids. In subsequent constructions, these would be substituted with CEG and conditionally rescued with the appropriate products (see below section). A comparison of the copy numbers in cells carrying single plasmid or dual-plasmid were carried out (
To further validate the compatibility between two artificial Ori sites where the loop sequences differed only by 2 bases, cells carrying two groups of plasmids differ only by the On sites were compared. Both groups had three plasmids with either kanamycin (Kan), chloramphenicol (Cam) or spectinomycin (Spec) resistant genes respectively. All the plasmids in the “Original group” (T7-ADS-ispA-Cam-p15A, T7-dxs-Kan-p15A and T7-ADS-ispA-Spec-p15A) had p15A as their Ori sites while the “Engineered group” (T7-ADS-ispA-Cam-p15AL2-5, T7-dxs-Kan-p15A and T7-ADS-ispA-Spec-p15AL2-8) were plasmids with different On sites (
Design of Conditional Host Cell System
As discussed above, a significant disadvantage of using plasmid is the potential loss during cell growth and this can be averted by selection pressure using antibiotics. Various approaches to maintaining single plasmids without the use of antibiotics in bacteria include the manipulation of essential genes such as dnpD, glyA, fabI, murA, acpP or the use of antidote/poison system. With multiple plasmids, however, the situation is more complex and will depend on the products to be manufactured. As yet, there has not been any demonstration of the use of these methods for the maintenance of multi-plasmid where multiple GOI are required to be simultaneously expressed and the plasmids stably maintained simultaneously.
A universal workflow to construct a multiple-plasmid/host system by generating auxotroph host and complementing with conditional essential genes (CEG) was developed. By knocking out multiple CEG in a host genome and at the same time placing them separately on various plasmids, only cells with all the essential components—the genome and all the plasmids with CEG will survive (
Not all metabolic enzymes are essential for growth. Hence, the knock-out of an enzyme in a pathway can be compensated by alternative biosynthetic pathways or the availability of isoenzymes, the existence of alternative enzymes and even broad specificity or multifunctional enzymes. Whether certain isozymes and alternative enzymes can complement for the missing one depends also on the expression levels and active states of the enzymes in that particular condition of growth.
The selection of CEG was not easily predicted from current knowledge and available resources. These databases contained collections of single but not multiple genes acting as conditionally or totally essential for growth in certain conditions. However, with the CAMPS knock-out of multiple CEG allowed the insertions and rescue of growth by using multiple plasmids with complementing CEG. Multiple CEG (e.g., metabolic enzymes) were knocked-out for performance of the strain in both growth and productivity.
With multiple co-existing compatible plasmids, auxotrophy could be achieved by using multiple CEG in series or in parallel of metabolic pathways. The criteria for the selection of either of these two approaches may be guided by a priori knowledge but was empirically established to provide a flexible, convenient and robust plasmid based platform for metabolic engineering and synthetic biology.
In order to conveniently introduce large numbers of CAMPS plasmids, sets of one to three CEG were distributed into subgroups based on their functionalities. For each subgroup, instead of using the complex rich medium, the auxotroph was rescued by the supplementation of specific metabolite that is economic and accessible to the cell. As a result, multiple CAMPS plasmids were sequentially or separately introduced into corresponding complementary host as subgroups, increasing the flexibility of pathway engineering.
To demonstrate the use of an enzyme in a serial biochemical pathway, three CEG from a pool of 94 genes that were thought to be essential in both glycerol and glucose minimum mediums but not in rich medium were selected and separately placed into three plasmids. The plasmids with these CEG (ΔaroA, ΔaroB and ΔaroC) were then shown to be stably maintained in single-plasmid/host, dual-plasmid/host and triple-plasmid/host systems.
Construction of Antibiotic-Free Multiple-Plasmid/Host System
To demonstrate the proposed work flow for the establishment of multiple-plasmid/host system (
The host and plasmid combinations were then processed for survival test. The strains harboring various plasmids were initially generated in rich medium with the addition of antibiotics. If there was no observation of growth after 90 hour incubation at 37° C. with shaking in the specified medium without antibiotics, the strain was considered to be non-viable. Based on the survival test, all the single knockout strains (MG1655 (DE3, ΔaroA); MG1655 (DE3, ΔaroB) and MG1655 (DE3, ΔaroC)) did not grow in minimum medium with either glycerol or glucose as carbon source confirming their essentialness in minimum medium (Table 2). On the other hand, the growth was only rescued by supplying the relevant plasmid such as inserting T7-ADS-Cam-aroB-p15A plasmid expressing aroB into MG1655 (DE3, ΔaroB) strain. At the same time, there was no growth of MG1655 (DE3, ΔaroB) strain with T7-ADS-Cam-p15A plasmid in minimum medium confirming that the rescue effects were due to the expressions of the CEG in the plasmid. No rescue activity was observed by the overexpression of irrelevant CEG by other plasmids confirming that the selected CEG could not serve as isoenzymes for each other. Similar observations were also observed in other multiple-plasmid systems (Table 3). In minimum medium, the MG1655, (DE3, ΔaroB) strain could only replicate when carrying both T7-ADS-Cam-aroB-p15A and T7-CYP450-CPR-Spec-aroC-pCL plasmids while the MG1655 (DE3, ΔaroABC) strain needed to carry all three plasmids (T7-dxs-Kan-aroA-pET, T7-ADS-Cam-aroB-p15A and T7-CYP450-CPR-Spec-aroC-pCL) for survival. Thus, these engineered hosts could only survive if they carried the relevant plasmids in minimum medium.
Extending the study, the plasmid copy numbers were measured after 2 day growth in minimum medium with no antibiotic selection. IPTG—the inducer for T7 promoter was supplied in some of the conditions to create selection pressure to cells harboring recombinant proteins expressed under the control of T7 promoter. For single-plasmid/host system, the T7-dxs-Kan-aroA-pET plasmid expressing E. coli native enzyme (dxs—1-deoxyxylulose-5-phosphate synthase) and T7-ADS-Cam-aroB-p15A plasmid expressing a plant gene (ADS—amorphadiene synthase) could be retained in both native strain (MG1655 (DE3)) and the modified strain (MG1655 (DE3, ΔaroA) or MG1655 (DE3, ΔaroB)), where induction of gene expression by IPTG were carried out. However, after induction (0.1mM IPTG) both plasmids were lost in the native strain but well retained in the modified strains (
Demonstration of the Use of CEG from Other Pathways
Three CEG (aroA, aroB and aroC,
Moreover, the multiple-knockout strains without aroA, aroB, araC genes and pdxH, pyrF, or proC gene in the genome could only survive when all four plasmids carrying necessary CEG were present (Table 4, strain 5-6). And the strain where the argBCH operon encoding three CEG in the biosynthetic pathway of arginine was knocked out can only grow in the presence of all the three CAMPS plasmids—T7-ADS-ispA-Cam-argB-p15AL2-10, T7-ADS-ispA-Cam-argC-p15AL2-6 and T7-ADS-ispA-Cam-argH-p15AL2-11 (Table 4, strain 4). These results lend further evidence that the multiple antibiotic-free plasmids expressing the CEG complemented the host where these genes were deleted.
Introduction of Multiple CEG Carrying Plasmids into Engineered Host
The construction of multiple CEG knockout host for antibiotic-free system can be achieved with the procedures described in
To overcome this, an antibiotic-free approach was proposed for the assembly of CAMPS (
Firstly, the growth properties of CEG knockout strains in various modified minimum mediums were tested. By supplying the direct product of CEG to the minimum medium with glucose as carbon source (MMG) (
Next, the studies on the use of antibiotic-free procedures of introducing multiple CEG carrying plasmids were designed and tested (Table 6). For MG1655 (DE3, ΔaroABC) strain where all the selected CEG were in a linear pathway, the modified strains with T7-dxs-Kan-aroA-pET and T7-CYP450-CPR-Spec-aroC-pCL plasmids were firstly transformed into cells grown in the MMG+SK medium where aroB was non-essential. The cells were then further transformed with the T7-ADS-Cam-aroB-p15A plasmid and selected in MMG medium (Table 6, strain 1).
Similarly, MG1655 (DE3, AargBCH) strain was generated by introducing T7-ADS-ispA-Cam-argH-p15AL2-11 plasmid in the first step in cells grown in the MMG+OR medium. Then the T7-ADS-ispA-Cam-argB-p15AL2-10 and T7-ADS-ispA-Cam-argC-p15AL2-6 plasmids were introduced into cells grown in MMG medium (Table 6, strain 5). For quadruple CEG knockout strains: MG1655 (DE3, ΔaroABC, ΔproC) strain (Table 6, strain 2), MG1655 (DE3, ΔaroABC, ΔpdxH) strain (Table 6, strain 3), MG1655 (DE3, ΔaroABC, ΔpyrF) strain (Table 6, strain 4), three plasmids carrying aroA, aroB or aroC genes were first introduced using protocols similar to the assembly of MG1655 (DE3, ΔaroABC) strain (strain 1) while additional product (PRO, PLP or UMP) was supplied to the relevant mediums for each of the strain. Using MMG medium for selection, the last plasmid (T7-ADS-ispA-Cam-proC-p15AL2-4, T7-ADS-ispA-Cam-pdxH-p15AL2-9 or T7-ADS-ispA-Cam-pyrF-p15AL2-1) was then introduced into the corresponding strain in the third step. By combining the procedures of stain 1 and strain 5, the assembly protocol for sextuple CEG knockout strain: MG1655 (DE3, ΔaroABC, ΔargBCH) strain was similarly carried out (Table 6, strain 6) where ARG was supplied to make argB, argC and argH genes non-essential while transforming the aroA, aroB, aroC carrying plasmids. The results of these studies demonstrated the success in introducing multiple plasmids with a variety of CEG by using minimal medium supplemented with the appropriate chemicals which were products of the CEG activities.
CAMPS for Metabolite Production
To demonstrate the utility of CAMPS, the production of amorphadiene through mevalonate (MVA) pathway was examined. The genes encoding the pathway enzymes were divided into three modules: the SAR module (hmgS, hmgR, atoB), the KKDI module (MVK, PMVK, MVD, idi) and the AA module (ADS, ispA). The modules were then separately placed into two sets of CAMPS plasmids: the MVA-213 set (TM2-SAR-Spec-aroC-p15A-1, TM1-KKID-Cam-aroB-p15A-8, TM3-AA-Kan-aroA-p15A) and the MVA-323 set (TM2-SAR-Spec-aroC-p15A-1, TM1-KKID-Cam-aroB-p15A-8, TM3-AA-Kan-aroA-p15A) where they were under the control of T7 promoter mutants of different strengths of controlling transcription (
Firstly, plasmid stability was tested for these strains when cultured in antibiotic-free minimum medium with glucose as the carbon source (
The production of amorphadiene were then compared when the two sets of plasmids were separately transformed into either the native strain (MG1655 (DE3)) or the engineered strain (MG1655 (DE3, ΔaroABC)). The strains were initially cultured in rich medium (2xPY++ medium) supplemented with three antibiotics (kanamycin, chloramphenicol and spectinomycin) to force the maintaining of all the plasmids. Although the MVA-213 set yielded less amorphadiene than the MVA-323 set as predicted, there was no difference between two strains with or without CAMPS (
Next, the productivity of the strains when cultured in antibiotic-free minimum medium with glucose or glycerol as the carbon source and various IPTG inductions were compared (
Discussion
For metabolic engineering and synthetic biology, a stable multiple-plasmid system is needed. As compatibility is an important issue when using multiple of plasmids simultaneously, an option is to use plasmids from different compatibility groups which are limited in numbers and also highly variable in copy numbers. The mechanisms of plasmid replication were discovered decades ago. In p15A Ori, the regulation of plasmid copy number and the compatibility of different plasmids are high related. A key step in controlling plasmid copy number is the inhibition of plasmid replication by RNA I which recognizes a complementary stem-loop structure of RNA II—the RNA primer that initiates the plasmid replication, Based on the mechanism, various attempts had been made to engineer the copy number of a plasmid by manipulating the sequences in the stem-loop structures with good success while attention to the compatibility issue of these modified plasmids has yet to be determined. In nature, plasmids with slight different sequences in the stem-loop structures are known to be compatible and are thought to evolutionarily related. For example, the p15A like plasmids: p15A, Col E1, RSF 1030 and CloDF13 are compatible with each other to a certain degree (
As described herein, the loop sequences of RNA I/II were specifically engineered and the modified plasmids were proven to be compatible. Plasmids with loop sequences differing by as few as two bases were found to be compatible. From these observations, an unlimited number of compatible plasmids can be created. In addition, by engineering the sequences in the origin of replication, these plasmids have varying copy numbers when present in the host. To engineer metabolic pathways, other than selecting high copy number plasmids, plasmids with similar copy numbers to the parental plasmid (p15A Ori) can be selected for the ease of control. Another important differentiating factor in this study as compared to the use of different naturally compatible plasmids is that the modified plasmid library generated here share the same mechanisms of replication and resources.
To expand the plasmid library, compatible plasmids can be generated by engineering the 2nd as well as the 1st and 3rd loop of p15A series Ori. Other series of plasmid family with replication mechanisms controlled by the recognition between RNA loops can be also engineered similarly. An example is the IncIα plasmid family whereas Inc RNA regulates the repZ translation and Inc18 plasmid family whereby RNA III inhibits the transcriptional of RepR protein.
Another challenge of using multiple plasmids is the maintenance of the plasmids inside the host during large scale fermentation processes. The use of antibiotics is not only limited by the lack of different antibiotics available but also challenges the purification, regulatory and cost issues. There are several antibiotic-free systems for the maintenance of a single plasmid usually for the purpose of recombinant production. However, there is yet to be an example of a robust antibiotic-free multiple-plasmid system.
Described herein is the development of compositions and methods to construct a versatile antibiotic-free multiple-plasmid system in which the plasmids carried genes that complemented the auxotroph host thereby stably maintaining the plasmids even under conditions of cellular stress. The first challenge was to construct strains with multiple essential gene knockouts, which could not survive in the absence of the appropriate plasmids. To overcome this, the choice of the conditional essential genes (CEG) allowed the strains to be conveniently created in rich medium where those genes are conditionally non-essential. Nine CEG from various biosynthetic pathways were experimentally demonstrated separately or in combinations. It was shown that the plasmids were stable even when the cells were subject to high selection pressure. After strain construction, another challenge was the lack of an antibiotic-free approach to introduce multiple plasmids into the strain because the limitation in the efficiency of co-transformation of plasmids to E. coli. This issue was surmounted with the use of modified growth medium where the strains lacking certain CEG could be grown by supplementing with the appropriate products. As a result, all the plasmids can be stepwise transformed in an iterative manner using respective modified growth medium at each step.
With two features: compatible plasmids using the same replication mechanism and culture in antibiotic-free medium, this novel multi-plasmid system is beneficial to studies involving the simultaneous manipulation of large number of genes in industrial large scale production process. As a proof-of-concept, this was demonstrated by the production of amorphadiene where the native host yielded much less amorphadiene as compared to CAMPS strains in antibiotic free environment.
Methods
Chemical, Growth Medium and Bacteria Strain
Unless stated otherwise, all chemicals were purchased from either Sigma or Merck. The yeast extract and peptone were purchased from BD. The growth medium was prepared supplying 20 g/L glucose or glycerol as carbon source. The 2xPY medium contained: peptone (20 g/L), yeast extract (10 g/L) and NaCl (10 g/L). The 2xPY++ medium contained: peptone (20 g/L), yeast extract (10 g/L), NaCl (10 g/L), glucose (20 g/L), Tween 80 (0.5%), HEPES (50 mM) and was the rich medium used for amorphadiene production. X110-Gold (Stratagene) or DH5a (Invitrogen) strain was used for plasmid construction, Unless stated otherwise, all the cells were grown at 37° C. with shaking (250 rpm). In certain conditions, various kinds of antibiotics were supplied as following: ampicillin—100 mg/L, kanamycin—50 mg/L, chloramphenicol—34 mg/L, spectinomycin—100 mg/L. MG1655 (DE3) strain was the same as the one used in previous study [47] and was the strain used for studies involving the construction and characterization of novel compatible plasmids. Cell density (absorbance at 600 nm) was measured by SpectraMax 190 microplate reader.
Amorphadiene Production
For amorphadiene production experiment, 800 μL of cells were cultured together with 200 μL of dodecane phase and cultured at 28° C. with shaking (250 RPM) in 15 ml FalconTM tube. The experiments were carried out for two days for rich medium (2xPY++ medium) and for four days for growth medium (growth medium with glycerol or glucose). Amorphadiene was trapped in the dodecane phase and quantified as previously described [35]. The dodecane phase was diluted 100 times in ethyl acetate and the amorphadiene was quantified by Agilent 7890 gas chromatography/mass spectrometry (GC/MS) by scanning 189 and 204 m/z ions, using trans-caryophyllene as standard. The amorphadiene concentrations were adjusted to the volume of cell suspension (0.8 ml) for report.
Strain Construction
Based on the parental strain (MG1655 (DE3)), the knock-out strains were constructed using the method described in the paper [48]. The primer pairs KO-aroAF/KO-aroAR, KO-aroBF/KO-aroBR, KO-aroCF/KO-aroCR, KO-pdxHF/KO-pdxHR, KO-proCF/KO-proCR and KO-pyrFF/KO-pyrFR were used to knock out the aroA, aroB, aroC, pdxH, proC, and pyrF genes receptively. The KO-argBCHF/KO-argBCHR primer pairs were used to knockout the argBCH operon consisting of argB, argC and argH genes. The knock-out strains were confirmed by PCR analysis with the primers listed in section “Primers used to check the knockout strains”. The “in” primers were targeting at the sequences removed from the genome and “out” primers were targeting at the genome regions outside the removed sequences.
Plasmid Construction
The strains used in the study were listed in “Table 7”. The T7-ADS-ispA-Cam-p15A plasmid, T7-dxs-Kan-pET plasmid and T7-CYP450-CPR-Spec-pCL plasmid were from previous studies. All plasmid were constructed with CLIVA method and primers were listed in “Table 7”. The mutagenesis of 2nd loop of p15A Ori was carried by PCR amplification of T7-ADS-ispA-Cam-p15A plasmid with I-15ALoop2-F/I-15ALoop2-R degenerate primer pairs. The I-aroA-F/I-aroA-R, I-aroB-F/I-aroB-R and I-aroC-F/I-aroC-R primer pairs were used to amplify the aroA, aroB and aroC genes from the genome of MG1655 strain (ATCC) together with their RBS sequences. The genes were then inserted into plasmids at locations adjacent to the antibiotic resistant genes to form a polycistronic expression. The I-KAN(aroAr)f/I-KAN(aroAf)r, I-CAM(aroBr)f/I-CAM(aroBf)r and I-SPE(aroCr)f/I-SPE(aroCf)r primer pairs were used to amplify the vectors respectively.
Plasmid Copy Number Measurement
The copy number of plasmid was defined as the ratio of the copy of plasmid DNA and to the copy of the genomic DNA. The copy numbers were measured by quantitative PCR (qPCR) with a standard curve prepared using linearized plasmid DNA or PCR product (1-3 kb) containing the amplicon (80-120 bps). Typically, 5 μL of cells whose medium were removed by centrifugation were diluted in 100 μL of water. The mixture was then heated at 95° C. for 20 min to lyse the cells. The cell debris was then removed by mild centrifugation and the solution containing all the DNAs was dilute 20 times for qPCR. The qPCR reactions were carried out in 25 μl final volume containing 5 μl diluted DNA samples, 1× Xtensa Buffer (Bioworks), 200 nM of each primer, 2.5 mM MgCl2 and 0.75 U of iTaq DNA polymerase (iDNA). The reactions were analyzed using a BioRad iCycler 4™ Real-Time PCR Detection System (Bio-Rad) with SYBR Green I detection and the following protocol: an initial denaturation of 1 min at 95° C., followed by 40 cycles of 20 s at 95° C., 20 s at 60° C., and 20 s min at 72° C. A melt curve was then measured to check the melting temperature of the amplicon. For all the studies, technical duplicates or triplicates were carried out. Primers were listed in “Table 8-Primers used for plasmid copy number measurement”. The antibiotic resistant genes were measured to represent the amount of various plasmids with Cam-F/Cam-R, Kan-F/Kan-R and Spe-F/Spe-R primer pairs. For amorphadiene production study, the pathway genes were measured to represent the amount of various plasmids with hmgS-F/hmgS-R, MVK-F/MVK-R and ADS-F/ADS-R primer pairs. The cysG-F/cysG-R primer pair was used to measure the cysG gene which is one copy in the genome to represent the copy number of genomic DNA.
Articles such as “a”, “an”, “the” and the like, may mean one or more than one unless indicated to the contrary or otherwise evident from the context.
The phrase “and/or” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when used in a list of elements, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but optionally more than one, of list of elements, and, optionally, additional unlisted elements. Only terms clearly indicative to the contrary, such as “only one of” or “exactly one of” will refer to the inclusion of exactly one element of a number or list of elements. Thus claims that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present, employed in, or otherwise relevant to a given product or process unless indicated to the contrary. Embodiments are provided in which exactly one member of the group is present, employed in, or otherwise relevant to a given product or process. Embodiments are provided in which more than one, or all of the group members are present, employed in, or otherwise relevant to a given product or process. Any one or more claims may be amended to explicitly exclude any embodiment, aspect, feature, element, or characteristic, or any combination thereof. Any one or more claims may be amended to exclude any agent, composition, amount, dose, administration route, cell type, target, cellular marker, antigen, targeting moiety, or combination thereof.
Embodiments in which any one or more limitations, elements, clauses, descriptive terms, etc., of any claim (or relevant description from elsewhere in the specification) is introduced into another claim are provided. For example, a claim that is dependent on another claim may be modified to include one or more elements or limitations found in any other claim that is dependent on the same base claim. It is expressly contemplated that any amendment to a genus or generic claim may be applied to any species of the genus or any species claim that incorporates or depends on the generic claim.
Where a claim recites a composition, methods of using the composition as disclosed herein are provided, and methods of making the composition according to any of the methods of making disclosed herein are provided. Where a claim recites a method, a composition for performing the method is provided. Where elements are presented as lists or groups, each subgroup is also disclosed. It should also be understood that, in general, where embodiments or aspects is/are referred to herein as comprising particular element(s), feature(s), agent(s), substance(s), step(s), etc., (or combinations thereof), certain embodiments or aspects may consist of, or consist essentially of, such element(s), feature(s), agent(s), substance(s), step(s), etc. (or combinations thereof). It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2014/000556 | 11/26/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61910617 | Dec 2013 | US |
