Modulation of ColE1-like plasmid replication for recombinant gene expression

Abstract

ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression.

Fig. (1). Overview of ColE1 plasmid homeostasis

ColE1 plasmid replication is controlled by a ~550bp sequence element known as plasmid origin of replication (ori). Plasmid replication: transcription of ori sequence off of the P2 promoter generates a primer RNA that primes leader-strand synthesis. An early (~100 bp) transcript forms three stem-loops with 6 or 7 unpaired bases at the end (SL1, 2, 3). Continued transcription leads to be formation of a new, larger stem-loop (SL4) through the interaction of two complementary sequence segments, α and β. This stem-loop is necessary for stable RNA-DNA hybrid (R-loop) formation. The formation of this hybrid is guided by a G-rich stretch of the primer that hybridizes with the complementary C-rich sequence on the template. The primer is processed by RNAse H to form a 3’-OH end and extended by DNA polymerase I. R-loop formation is the only absolute requirement for plasmid replication initiation. Antisense regulation: Plasmid copy number is regulated by an antisense RNA whose expression is under the control of the P1 promoter. This inhibitor RNA forms three stem-loops, which are complementary to SL1,2,3 of the pre-primer. The interaction between the tips of these loops forms a transient “kissing complex” and then a permanent hybrid, a process that is nucleated by the antitail portion of the transcript at the 5’ end. This hybrid favors de interaction of the β segment of sequence with another stretch of sequence (γ), preventing the formation of SL4 and therefore of R-loop formation. The levels of antisense transcript are proportional to the number of plasmids, creating a negative feedback loop that keeps plasmid copy number constant for a given set of conditions. RNAP=RNA polymerase

Fig. (2). Functional determinants of ColE1 plasmid origins of replication and examples of loss-of-function mutations

The sequence of the ColE1 ori is presented as originally described [84]. Functional elements are highlighted as boxes and secondary structures are underlined. Of these, three elements are conserved, suggesting common functionality between ColE1, pMB1, p15A, RSF1030, and CloDF13: 1) stem-loops 1, 2, and 3, although the unpaired sequences at the tip show divergence (for antisense regulation) [6]; 2) the potential to expose an anti-tail (for the formation of a stable RNAI/RNAII complex) [53]; 3) G-rich and C-rich stretches (whose interaction is likely involved in preventing the release of the nascent transcript) [9]. The RNA primer hybridizes with a ~30 nucleotide stretch around the DNA/RNA switch. Beyond 7 nucleotides downstream of the RNA/DNA switch, sequence homology within ColE1 family plasmids is lost, except for positions +110 and +170 [6], which contains a putative binding site for the PriA [85]. PriA is normally recruited to restart replication at sites of replication fork collapse (reviewed in [86]). It seems that plasmids have co-opted this mechanism of DNA repair to complete the replication of their own genomes (reviewed in [85]). In agreement with their relative lack of conservation, sequences downstream of position +14 are dispensable [87]. Mutations that decrease the efficiency of replication initiation in ColE1 or in the closely related pMB1 ori are indicated and numbered. Mutations isolated together in combination are joined with a line. The reference key for loss of function mutations is presented separately in Table (1), which lists phenotype, references and second-site suppressors for each mutant.

Fig. (3). Metabolic response to amino acid starvation

a. Stringent response. Amino acid depletion causes ribosome idling, which is sensed by a ribosomal protein, ppGpp-synthetase I (encoded by relA). Rel A activation produces an alarmon, (p)ppGpp, which interacts with RNA Polymerase. Conformational changes in the RNA polymerase lead to changes in promoter specificity, reducing the synthesis of stable RNAs and increasing expression of biosynthetic pathways (reviewed in [32]). This leads to a marked suppression in protein synthesis. b Relaxed response. RelA strains, which are defective in ppGpp synthetase I, maintain low levels of (p)ppGpp under conditions of amino acid starvation. This maintains the level of protein synthesis to continue, but generates high levels of uncharged tRNAs, which eventually also have an impact on levels of translation.

Fig. (4). Models of disregulation of plasmid replication caused by accumulation of uncharged tRNAs in relA stains

a Formation of codon-anticodon complexes with tRNA. A structural similarity and >40% sequence homology was noticed between SL1, 2, and 3 of RNA I, II or both and the cloverleaf structure of t-RNAs. Yavachev et al. postulated that competitive hybridization between the anticodon loop of tRNA and the corresponding anticodon-like loops of RNA I or RNA II could interfere with the formation of RNAI/RNAII hybrids [36]. This model predicts that changes in plasmid copy number with deprivation of individual amino acids should correlate with the homology between the corresponding tRNAs and loops in RNA I and II. Note that each unpaired loop provides three different options for hybridization with tRNA anticodon sequences. A 7-nt loop is shown in the inset as an example: centered (boxed), shifted (continuous line) and very shifted (broken line). Based on solvent exposure, centered interactions are assumed to be the preferred ones, while the very shifted ones would be the least preferred ones. b. CAA-OH tRNA hybridization with UGG sequence of RNAI or RNA II. 3’-CAA of uncharged tRNAs hybridizes with the GGU motif of either RNA I or RNAII (depending on the specific sequence of each ori), and this bond is stabilized because a proton given by the CCA is trapped by an electron hole (GG+) at RNA I, RNA II loops [37]. The specific effects of individual amino acid deprivation are explained because in ColE1 plasmids, the GGU…G sequence is encoded in RNA I, which is the more abundant of the two ori transcripts and therefore sensitive to the levels of uncharged tRNA, which in turn corresponds to the relative abundance of different amino acids in E. coli proteins. This model predicts that starvation for any amino acid should lead to runaway plasmid replication in the case of PIGDM1, CloDF13 and other members of the ColE1/pMB1 family of enzymes that encode the GGU…G in the RNAII transcript. Note that both models propose a role of tRNAs in regulation of replication initiation. It has been proposed that tRNAs originated from RNA replication primers not unlike RNA II, as clover leaf-like structures frequently prime replication of viral and plasmid genomes (note the clover leaf-like structure of RNA II) [88].

Fig. (4). Models of disregulation of plasmid replication caused by accumulation of uncharged tRNAs in relA stains

a Formation of codon-anticodon complexes with tRNA. A structural similarity and >40% sequence homology was noticed between SL1, 2, and 3 of RNA I, II or both and the cloverleaf structure of t-RNAs. Yavachev et al. postulated that competitive hybridization between the anticodon loop of tRNA and the corresponding anticodon-like loops of RNA I or RNA II could interfere with the formation of RNAI/RNAII hybrids [36]. This model predicts that changes in plasmid copy number with deprivation of individual amino acids should correlate with the homology between the corresponding tRNAs and loops in RNA I and II. Note that each unpaired loop provides three different options for hybridization with tRNA anticodon sequences. A 7-nt loop is shown in the inset as an example: centered (boxed), shifted (continuous line) and very shifted (broken line). Based on solvent exposure, centered interactions are assumed to be the preferred ones, while the very shifted ones would be the least preferred ones. b. CAA-OH tRNA hybridization with UGG sequence of RNAI or RNA II. 3’-CAA of uncharged tRNAs hybridizes with the GGU motif of either RNA I or RNAII (depending on the specific sequence of each ori), and this bond is stabilized because a proton given by the CCA is trapped by an electron hole (GG+) at RNA I, RNA II loops [37]. The specific effects of individual amino acid deprivation are explained because in ColE1 plasmids, the GGU…G sequence is encoded in RNA I, which is the more abundant of the two ori transcripts and therefore sensitive to the levels of uncharged tRNA, which in turn corresponds to the relative abundance of different amino acids in E. coli proteins. This model predicts that starvation for any amino acid should lead to runaway plasmid replication in the case of PIGDM1, CloDF13 and other members of the ColE1/pMB1 family of enzymes that encode the GGU…G in the RNAII transcript. Note that both models propose a role of tRNAs in regulation of replication initiation. It has been proposed that tRNAs originated from RNA replication primers not unlike RNA II, as clover leaf-like structures frequently prime replication of viral and plasmid genomes (note the clover leaf-like structure of RNA II) [88].

Fig. (5). Examples of gain-of-function mutations

Mutations that increase plasmid copy number in ColE1 or in the closely related pMB1 ori are indicated and numbered on the sequence of ColE1 originally described in [84]. Mutations isolated together in combination are joined with a line. The reference key for the mutations is presented separately in Table (2), which lists phenotype and references for each mutant.

Fig. (6). Complex proprietary mutants

a Deletion mutants in SL1, 2 and 3 [49, 50]. Deleted sections as reported in [50] (and patented [49]) are represented as lines below the sequence, with their identifiers next to them. For comparison, point mutants that increase copy number are also presented as numbers above the sequence. The legend to the numbers can be found in Table (2). b Inverted homology to tRNAs. Seven bases of SL2 (all but one G, in bold) were replaced by their complement bases, thereby inverting tRNA homologies without changing the complementarity between inhibitor and target RNA or any structural features of the stem-loop. A construct bearing the mutant ori and superoxide dismutase showed no signs of disregulation after induction of the recombinant protein in batch fermentation culture.

Fig. (7). Effect of mutations affecting SL1, 2 ,or 3 interactions on contralateral regulation

A G→ A point mutation is presented as an example. If the mutant plasmid coexists with the wild-type plasmid, two inhibitor RNA I species are present. The corresponding contralateral interactions are: WT RNA I (encoding C) is opposite an A, a weak interaction, and mutant RNA I (encoding a U) opposite a G, a less-destabilizing interaction. This asymmetrical effect leads to the preferential elimination of the plasmid that is more efficiently repressed, the wild-type plasmid in this case.