Patent Application
20250043293
This invention provides materials and methods allowing expression of RNA and proteins in Bacillus, especially B. subtilis and similar species, with a new tightly regulated, integrative and inducible expression system.
Bacillus subtilis is a model organism for studies of Gram-positive bacterial biology and systems biology of cellular differentiation, stress responses, and multicellular organization. Additionally, B. subtilis is among the most widely used hosts for protein production in the biotechnology industry due to its ability to secrete proteins into the cell medium, its non-pathogenic GRAS (generally recognized as safe) designation, and its high genetic tractability. For example, B. subtilis is used for large-scale production of lipases, proteases, and amylases, among other industrially-relevant proteins.
However, B. subtilis work has been limited by a lack of high-quality synthetic biological parts. For example, biotechnology applications require inducible promoters capable of switching between a low production state for early-stage culturing and a high production state that maximizes protein yield during fermentation. Such parts are particularly important when the recombinant protein or metabolic pathway of interest are toxic to the host cells when overproduced.
Typical inducible promoters in B. subtilis have dynamic ranges (ratio of output protein expression in the presence versus absence of inducer) of at most a few hundred. While a B. subtilis bacitracin-inducible promoter with a 1,000-fold range has been reported (13), it requires antibiotic selection to maintain a multicopy plasmid, and its activity is transient and shuts down less than two hours after induction, likely due to an endogenous bacitracin stress response. In contrast, for the Gram-negative bacterium Escherichia coli, inducible promoters have been engineered with dynamic ranges greater than 1,000 or even 10,000.
Thus, improved B. subtilis inducible promoter systems are greatly needed in the art. The ideal system will be very tightly regulated, result in high levels of protein expression when induced, have 10,000 or even greater fold dynamic range, and ideally be integratable into the genome.
Protein expression systems that are activated in response to chemicals such as IPTG, xylose or bacitracin currently exist for B. subtilis. However, their fold-activation values are fairly low, and their active output levels are limited by the B. subtilis endogenous transcription machinery. On the other hand, a few systems based on the exogenous viral T7 RNA Polymerase (T7 RNAP) have been previously built. While these achieve high expression levels, protein production in the inactive state or “leakiness” is fairly high and therefore fold-activation is still low.
Here, we have engineered an extraordinarily stringent and strongly inducible protein expression system for Bacillus. This system combines the viral T7 RNAP, which produces high amounts of recombinant protein in the active state, with the LacI bacterial transcription factor that inhibits production of both the protein and the viral polymerase, resulting in nearly undetectable output in the inactive state. Our system achieves greater than 10,000-fold or even 20,000-fold activation in response to a chemical inducer. We expect this system to improve yield of existing protein production, especially toxic proteins, or metabolic engineering applications using B. subtilis and possibly other Bacillus species, and enable synthesis of new proteins and chemicals that were previously unfeasible.
Our LacI-T7 inducible system comprises the following elements:
For an integrative sequence, these elements should be flanked by first and second portions of a non-essential portion of the Bacillus genome, thereby allowing integration into that genome. The order of parts is typically not critical, but whatever the order, the cassette is flanked by the integrating sequences such that everything there between is integrated into the genome. In addition, sequences to optimize expression of the target ORF can also be included, such as ribosome binding sites, enhancers, terminators, and the like.
The cassette can be assembled and used as is, or can be formulated as an expression vector—capable of self-reproduction and ORF expression. For an expression vector, additional vector sequences are added, allowing replication in Bacillus and/or E. coli (e.g., ori), marker genes for selection (e.g., antibiotic resistance), unique restriction endonuclease (RE) sites or the multi-cloning site, shuttle sequences allowing the vector to shuttle e.g., between E. coli and Bacillus (both ori sequences), or between yeast and Bacillus (Bacillus ori and autonomously replicating sequence (ARS), a yeast centromere (CEN), and a yeast selectable marker), viral packaging sequences, protein degradation tags, and the like. It may also be possible to formulate the cassette described herein as minivectors, such as those described by Twister® (Houston TX).
LacI and the Phy-spank promoter were obtained from pDR111 (ATCC® 53123), a commercially available plasmid for genomic integration in B. subtilis. The PT7lac promoter sequence is identical to that included in some of the commercially-available E. coli pET plasmids, and was synthesized via oligo annealing and extension. The sequence of the T7 RNAP is identical to that found in the E. coli BL21 (DE3) strain, and was obtained from an E. coli plasmid.
DNA coding for these elements was assembled, together with a spectinomycin resistance cassette. Flanking sequences for genome integration were added that were homologous to the B. subtilis amyE genomic locus, but any non-essential loci could be used. The assembled DNA cassette was then integrated into the Bacillus genome via standard Bacillus transformation methods.
In the inactive state, LacI repression of both T7 RNAP production and PT7lac ensures very low production of recombinant protein. Upon addition of chemical inducer IPTG to the cell media, LacI-repression is relieved and the T7 RNAP is produced. The T7 RNAP can now transcribe the recombinant gene from the now unrepressed PT7lac promoter.
To assess the performance of the LacI-T7 system, we placed superfolder GFP (sfGFP) under PT7lac and measured cell fluorescence in the presence or absence of IPTG. This resulted in high levels of fluorescence in the presence of IPTG, but undetectable levels in its absence. Based on estimates of the limit of detection of our flow cytometer instrument, we placed a lower bound on the fold-activation of this system at 20,000. We have also tested the system with other report
The invention includes any one or more of the following in any combination(s) thereof:
subtilis;
Escherichia or yeast.
As used herein “recombinant” or “engineered” is relating to, derived from, or containing genetically engineered material. In other words, the genome was intentionally manipulated by humans in some way.
“Expression vectors” are used in accordance with the art-accepted definition of a plasmid, virus, cosmid, or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expression vectors also exist and either can be used.
As used herein, “inducible” means that gene expression can be controlled by the hand-of-man, by adding e.g., a ligand to induce expression from an inducible promoter. Exemplary inducible promoters include the lac promoter, inducible by isopropylthio-β-D-galactopyranoside (IPTG), the yeast AOX1 promoter inducible with methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters. “Constitutive” means there is always expression from that promoter. Pveg is an example of same.
As used herein, “integrative” or “integratable” means the nucleic acid has the ability to integrate into a B. subtilis or equivalent genome.
As used herein, an “integrated sequence” means the sequence has been integrated into the host genome, as opposed to being maintained on an expression vector or as a separate integration cassette. It will still be expressible, either inducibly or constitutively. Herein, we are more interested in tightly regulated and highly activatable inducible expression vectors and/or cassettes that are integrative or are integrated.
As used herein, “operatively connected” or “operatively coupled” with respect to DNA sequences means that the oligonucleotide segments are connected in such a way as to allow the DNA (or RNA or protein derived therefrom) to be functional in a cell. Typically, this means e.g., the correct spacing, essential regulatory sequences, and reading frame (if applicable) are maintained.
As used herein, “homolog” means an enzyme with at least 40% identity to one of the listed sequences and also having the same general catalytic activity, although kinetic parameters of the reactions can of course vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it is typical for bacterial sequences to diverge significantly (40-60% identity), yet still be identifiable as homologs, while mammalian species tend to diverge much less (80-90% identity). Unless specified otherwise, any reference to an enzyme herein also includes its homologs that catalyze the same reaction.
As used herein, references to cells or bacteria or strains and all such similar designations include progeny thereof. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations that have been added to the parent. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.
The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.
The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.
The phrase “consisting of” is closed, and excludes all additional elements.
The phrase “consisting essentially of” excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention.
The following abbreviations are used herein:
licheniformis.
Table 1. Data.
Table 2. Various sequences SEQ ID NO 10-22.
In more detail, we have engineered a stringent (i.e. non-leaky) and highly-inducible LacI-T7 promoter system for B. subtilis. Our system utilizes the hybrid PT7lac promoter to express a gene of interest, and the IPTG-inducible promoter Phy-spank to express the T7 RNA Polymerase (T7 RNAP) (
In the absence of the IPTG inducer, the repressor lacI is active and both T7 RNAP and PT7lac are thereby repressed. This dual repression minimizes leaky expression of the gene of interest. Thus, the system is very stringent, with little to no detectable leaking. Upon addition of IPTG, LacI activity is inhibited, and the newly produced T7 RNAP strongly transcribes the gene of interest from the now de-repressed PT7lac (
Our LacI-T7 design is conceptually similar to some variants of the commercial E. coli pET expression system, where IPTG also induces both expression of T7 RNAP and de-repression of PT7lac. However, our system uses Bacillus or B. subtilis-specific promoters (other than PT7lac) and ribosome-binding sites (RBSs). Additionally, while a few B. subtilis gene expression systems based on T7 RNAP have been previously reported, they use the LacI-independent Pr7 instead of PT7lac, resulting in dynamic ranges of less than 50.
To validate our design, we placed a sfgfp reporter gene22 with a codon-optimized N-terminal fragment (sfgfp*) under control of LacI-T7, the self-cleaving ribozyme RiboJ, and RBS MF001, and integrated it into the non-essential amyE locus of the B. subtilis genome as a single copy (
We utilized flow cytometry to characterize sfGFP fluorescence levels in the absence and presence of IPTG. We found that, in the absence of inducer, sfGFP fluorescence equals 17.9±4.7 molecules of equivalent fluorescein (MEFL) (
To validate that this high dynamic range is preserved when expressing a different gene of interest, we placed the lacZ reporter gene under LacI-T7 (
Phy-spank is a variant of the IPTG-inducible promoter Pspac optimized for higher expression and dynamic range, and is perhaps the most widely used B. subtilis inducible promoter system. We constructed a second B. subtilis strain wherein sfgfp* was expressed under the control of Phy-spank, RiboJ, and RBS MF001 to compare its performance to that of LacI-T7. We found that Phy-spank exhibits much greater leakiness (565±18 MEFL) and a lower maximal output (160,000±14,000 MEFL) than LacI-T7, resulting in a dynamic range of only 282±18 (
Next, we measured the steady state transfer function of both systems by growing the corresponding strains under different concentrations of IPTG. In both cases, sfGFP fluorescence increases as a function of IPTG concentration in a manner well-approximated by a Hill function (
Finally, we characterized the response dynamics of both systems after an instantaneous addition of saturating IPTG (
The superior performance of LacI-T7 appears to arise from its unique design features. First, high maximal expression results from the use of T7 RNAP, a strong viral RNA polymerase which is capable of re-directing all bacterial resources towards expression of a single output gene. In contrast, previous expression systems based on endogenous promoters are limited by the native transcriptional machinery and are subject to competition with other endogenous promoters. Second, leaky expression in the absence of inducer is reduced via the dual repression activity of LacI. In E. coli, a similar design has been shown to reduce basal expression by more than an order of magnitude compared to an unmodified PT7 output promoter.
LacI-T7 should be useful in quantitative studies of B. subtilis biology. In particular, its stable single-copy chromosomal location and its low leakiness are desirable for analyses of ultrasensitive or excitable networks where low amounts of excess protein can cause cells to undergo dramatically different differentiation programs.
We also expect LacI-T7 to be useful for heterologous protein expression applications. High expression from PT7lac should enable high yields of both cytoplasmic and secreted proteins. Additionally, low leakiness in the absence of inducer should allow for fast initial cell growth, even with potentially toxic proteins. Furthermore, LacI-T7 is integrated into the B. subtilis genome, and thus will not suffer from plasmid instability issues or require strong selective pressure to be maintained. Because it relies on the orthogonal T7 polymerase, LacI-T7 could also be ported to other industrially-relevant Bacillus species or strains with little additional work.
All cloning and experiments were performed in B. subtilis strain PY79. Primers were ordered from Integrated DNA Technologies, Inc. Phy-spank was amplified from integration plasmid pDR111. PT7lac was constructed via oligo annealing and extension. Synthetic RBS MF001 was obtained from integration plasmid pMF35. Genomic homology fragments required for chromosomal integration were amplified from the purified genome of B. subtilis PY79.
All systems were built as linear double-stranded integration module (IM) or cassette, as we have previously described. Our IMs contain the DNA of interest and a selection marker flanked by 1.5 kb-long sequences homologous to the amyE locus of the B. subtilis genome where chromosomal integration via double crossover occurs. IMs were assembled from PCR-amplified parts using Golden Gate. The resulting Golden Gate product was amplified using NEB Phusion DNA Polymerase and gel purified to obtain the IM. 500 ng was transformed into competent B. subtilis using standard transformation methods.
The transformants were plated on selective media. Colonies were picked the next day and grown in LB media at 37° C. and 250 RPM for a few hours. Finally, freezer stocks were prepared with 700 μL culture and 300 μL 60% glycerol, and stored at −80° C. This method avoids sub-cloning of integration plasmids in E. coli, as long as enough PCR-amplified DNA can be obtained. The complete sequences of all IMs constructed in this study can be found in GenBank via the following accession numbers: Phy-spank-sfgfp: MN005205, LacI-T7-sfgfp: MN005204, LacI-T7-lacZ: MN005206.
For DNA sequence verification, an overnight LB culture was grown from a freezer stock, and 2 μL saturated culture was used as template for a 50 μL PCR reaction, either with Taq or Phusion DNA Polymerase. PCR products obtained in this fashion were gel-purified and sent for sequence verification to Genewiz, Inc.
We used a modified M9 medium for all experiments. 1 L 5×M9 salts at PH˜6.8 were prepared with 64 g Na2HPO4·7H2O, 15 g KH2PO4, 2.5 g NaCl, 5 g NH4Cl, 9.2 mL 6M HCl, and up to 1 L dH2O. For 1 L M9, we used 200 mL 5×M9 salts, 20 mL 10% casamino acids, 6.67 mL 60% glycerol, 1 mL 50 mM FeCl3/100 mM C6H8O7 solution, 2 mL 50 mM MnSO4, 2 mL 1M MgSO4, 100 μL 1M CaCl2, and dH2O up to 1 L.
For each experiment, an overnight LB culture was started from the freezer stock of each relevant strain. The next day, saturated cultures (OD600˜3) were diluted 105-fold in M9. Media was distributed in culture tubes (3 mL per tube), inoculated with the appropriate inducers (0 μM or 500 μM IPTG), and incubated in a shaker operating at 250 rpm and 37° C., until the OD600 reached between 0.08 and 0.15 (around 6 hours). Culture tubes were then transferred to ice. 100 μL of each sample was transferred to a flow cytometry tube containing 1 mL PBS for measurement.
The sfGFP fluorescence distribution of each culture was measured using a BD FACScan flow cytometer with an excitation source of 488 nm and an emission window of 510/21 nm. 30,000 events were collected per sample. A suspension of calibration beads (Spherotech® RCP-30-5A) in PBS was measured with each experiment. After data acquisition, raw .fcs flow cytometry files were processed using FlowCal.
Cell populations were gated by forward scatter/side scatter density (not shown) retaining 50% of the total number of events. Next, fluorescence of each gated event in arbitrary units was converted into standardized MEFL values using the calibration bead data. The total cellular fluorescence of each culture sample was then obtained by calculating the median MEFL fluorescence of all gated events in that sample. Finally, the reported sfGFP fluorescence values were obtained by subtracting the total cellular fluorescence of a wild-type PY79 sample measured the same day from each sample's total cellular fluorescence. Numerical sfGFP fluorescence values of every sample and replicate can be found in Table 1.
Codon optimization of the N-terminal sequence of the sfgfp ORF was performed to decrease secondary structure with the RBS and increase translation efficiency. To do so, for each of the first 15 codons of the original sfgfp sequence, a synonymous codon was chosen to reduce GC and increase AU content, with A preferred over U, with no regard for codon frequency. These changes were confirmed to increase the mRNA secondary structure free energy (and thus decrease secondary structure stability) via Nupack, by using the sequence from the transcription start site up to the 90th nucleotide residue of the ORF. The complete optimized sfgfp* sequence can be found in Table 2.
Statistical methods are not reproduced herein, but can be found in Castillo (2019).
Each of the following references is incorporated by reference herein in its entirety for all purposes:
This application claims priority to U.S. Ser. No. 62/776,132, filed Dec. 6, 2018, and incorporated by reference in its entirety for all purposes.
This invention was made with government support under Grant Nos: R21AI115014 and MCB1616755, awarded by the NIH and NSF, respectively. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/65080 | 12/6/2019 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62776132 | Dec 2018 | US |
