WO2022115636A1

WO2022115636A1 - Cell viability switch for genetically modified organisms

Info

Publication number: WO2022115636A1
Application number: PCT/US2021/060866
Authority: WO
Inventors: David James O'HAGAN
Original assignee: Esperovax Inc.
Priority date: 2020-11-25
Filing date: 2021-11-24
Publication date: 2022-06-02
Also published as: WO2022115636A9

Abstract

The disclosure provides compositions, microorganisms, and methods for producing heterologous proteins utilizing a genetic construct that comprises: (i) a nucleic acid comprising a removable coding sequence flanked on either end by recombination sites, wherein said removable coding sequence comprises at least one gene encoding a protein having a function essential to replication of the microorganism, and (ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, capable to delete the gene encoding the protein having a function essential to replication, and (iii) at least one nucleic acid comprising a gene encoding a heterologous protein. Deletion of the gene encoding the protein essential to replication improves production of the heterologous protein and reduces the risk of contaminating the environment with the genetically recombinant components. In embodiments, the removable coding sequence further comprises a gene encoding a cytotoxic agent capable to kill cells still capable of replication.

Description

CELL VIABILITY SWITCH FOR GENETICALLY MODIFIED ORGANISMS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to US Provisional Application No. 63/118,611, filed November 25, 2020, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

FIELD OF INVENTION

[0002] The present invention relates generally to compositions and methods for producing heterologous proteins in recombinant host cells. In some embodiments the methods include inhibiting cell replication during the induction phase of gene expression to produce a heterologous protein of interest. Inhibition of replication may improve production of heterologous proteins by reducing the metabolic burden of replication.

BACKGROUND OF THE INVENTION

[0003] Therapeutic proteins are extensively used in the treatment of disease. Because of the increasing prevalence of chronic diseases such as cancer, anemia, hemophilia, infectious diseases, and multiple sclerosis, the demand for vaccines, antibodies and other therapeutic proteins is high. In order to meet the increased demand for therapeutic proteins, compositions and methods for efficient production of therapeutic proteins is needed in the art. Fortunately, the following disclosure provides for this and other needs.

SUMMARY OF THE INVENTION

[0004] The present inventors have discovered a method for increasing cell metabolism during heterologous protein production while simultaneously preventing the host cell from replicating during and after heterologous protein production, thus reducing the risk of contaminating the environment with the genetically recombinant components. Furthermore, to ensure that any cells which maintain the ability to replicate do not escape into the environment, in some embodiments, the compositions and methods provide for expression of a gene encoding a cytotoxic agent operably linked to a regulatable promoter which can be activated exclusively in replication competent cells so as to eliminate the cells still capable of replication.

[0005] Therefore, in one aspect, the disclosure provides a genetic construct for producing one or more heterologous proteins in a host microorganism, wherein the construct comprises: (i) a nucleic acid comprising a removeable coding sequence flanked on either end by recombination sites oriented in the same direction wherein said removeable coding sequence comprises at least one gene encoding a protein having a function essential to replication of the microorganism, and

(ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to a regulatable promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the microorganism, thereby inhibiting cell replication, and

(iii) at least one nucleic acid comprising a gene encoding a heterologous protein, wherein the gene encoding the heterologous protein is operably linked to a regulatable promoter.

[0006] In one embodiment, the regulatable promoter operably linked to the gene encoding the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein are inducible and activated by the same inducer. In another embodiment the removeable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter, wherein the gene encoding a cytotoxic agent is downstream (3’) to the gene encoding a protein having a function essential to replication of the microorganism. In another embodiment, the regulatable promoter operably linked to the cytotoxic agent is different from the regulatable promoter operably linked to the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein. In another embodiment, the cytotoxic agent is a herpes simplex virus (HSV) thymidine kinase. In another embodiment, the regulatable promoter is a TEF1 promoter. In another embodiment, the host microorganism is selected from the group consisting of a yeast, a bacterium, and a cyanobacterium. In another embodiment, the host microorganism is a yeast. In another embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the gene encoding the protein having a function essential to replication of the host microorganism is an endogenous DNA polymerase. In another embodiment, the heterologous recombinase enzyme is Cre recombinase and the recombination sites are lox sites. In another embodiment, the regulatable promoter is a Gal 10 promoter. In another embodiment, the heterologous protein is selected from the group consisting of a hormone, an anti-inflammatory protein, a cell-wall permeabilizing agent, an immunogen, a virus like particle (VLP) forming protein sequence, preferably wherein said VLP-forming protein sequence comprises a viral structural protein or functional fragment thereof, wherein said viral structural protein comprises a matrix protein, a capsid protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, or combinations thereof. In another embodiment, the cell wall permeablizing agent is selected from the group consisting of a cell wall degrading enzyme, and an inhibitor of cell wall biosynthesis. In another embodiment, the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase. In another embodiment, the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

[0007] In another aspect, the disclosure provides a genetic construct for producing one or more heterologous proteins in a yeast cell, comprising:(i) a nucleic acid comprising a removeable coding sequence flanked on either end by recombination sites oriented in the same direction wherein said removeable coding sequence comprises an endogenous DNA polymerase, and a gene encoding a herpes simplex virus (HSV) thymidine kinase operably linked to a TEF1 promoter, wherein the gene encoding the HSV thymidine kinase is located adjacent to and downstream of the endogenous DNA polymerase, (ii) a nucleic acid comprising a heterologous Cre recombinase enzyme, wherein the heterologous Cre recombinase is operably linked to a Gal 10 promoter, and wherein the Cre recombinase is capable of acting on the lox recombination sites to recombine the lox recombination sites and delete the gene encoding the endogenous DNA polymerase, thereby inhibiting cell replication, (iii) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is a cell wall degrading enzyme operably linked to a Gal 10 promoter, and (iv) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is an immunogen operably linked to a Gal 10 promoter.

[0008] In one embodiment, the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase. In another embodiment, the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

[0009] In another aspect, the disclosure provides a method for producing a heterologous protein, the method comprising: (a) cultivating a host microorganism that comprises: (i) a nucleic acid comprising a removeable coding sequence flanked on either end by recombination sites oriented in the same direction, wherein said removeable coding sequence comprises at least one gene encoding a protein having a function essential to replication of the microorganism, and (ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to an inducible promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the microorganism, thereby inhibiting cell replication, and (iii) at least one nucleic acid comprising a gene encoding a heterologous protein, wherein the gene encoding the heterologous protein is operably linked to an inducible promoter, and (b) inducing the inducible promoter.

[0010] In one embodiment, the inducible promoter operably linked to the gene encoding the heterologous recombinase enzyme and the inducible promoter operably linked to the gene encoding the heterologous protein are activated by the same inducer. In another embodiment, the removeable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter, wherein the gene encoding a cytotoxic agent is downstream (3’) to the gene encoding a protein having a function essential to replication of the microorganism. In another embodiment, the regulatable promoter operably linked to the cytotoxic agent is different from the regulatable promoter operably linked to the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein. In another embodiment, the method further comprises the step of inducing the regulatable promoter operably linked to the cytotoxic agent, thereby inducing expression of the cytotoxic agent. In another embodiment, the cytotoxic agent acts on a precursor compound to convert the precursor compound to a toxic product. In another embodiment, the method further comprises providinga precursor compound to the host microorganism. In an embodiment, the providing of the precursor compound to the host microorganism occurs in vitro. In other embodiments, providing the compound to the host microorganism occurs in vivo , for example under conditions where the host microorganism is administered to a subject, the compound can also be administered to the subject such that the providing of the precursor compound to the host microorganism occurs in the subject. In another embodiment, the cytotoxic agent is a herpes simplex virus (HSV) thymidine kinase, the regulatable promoter is a TEF1 promoter, and the precursor compound is gancyclovir. In another embodiment, the host microorganism is selected from the group consisting of a yeast, a bacterium, and a cyanobacterium. In another embodiment, the host microorganism is a yeast. In another embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the gene encoding the protein having a function essential to replication of the host microorganism is an endogenous DNA polymerase. In another embodiment, the heterologous recombinase enzyme is Cre recombinase and the recombination sites are lox sites. In another embodiment, the inducible promoter is a Gal 10 promoter. In another embodiment, the heterologous protein is selected from the group consisting of a hormone, an anti-inflammatory protein, a cell-wall permeabilizing agent, an immunogen, a virus like particle (VLP) forming protein sequence, preferably wherein said VLP-forming protein sequence comprises a viral structural protein or functional fragment thereof, wherein said viral structural protein comprises a matrix protein, a capsid protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, or combinations thereof. In another embodiment, the cell wall permeablizing agent is selected from the group consisting of a cell wall degrading enzyme, and an inhibitor of cell wall biosynthesis. In another embodiment, the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase. In another embodiment, the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

[0011] In another aspect the disclosure provides a method for producing a vaccine, the method comprising: (a) cultivating a yeast cell, that comprises: (i) a nucleic acid comprising a removeable coding sequence flanked on either end by recombination sites oriented in the same direction, wherein said removeable coding sequence comprises an endogenous DNA polymerase, and a gene encoding a herpes simplex virus (HSV) thymidine kinase operably linked to a TEF1 promoter, wherein the gene encoding the HSV thymidine kinase is located adjacent to and downstream of the endogenous DNA polymerase, and (ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to an Gal 10 promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the microorganism, thereby inhibiting cell replication, (iii) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is a cell wall degrading enzyme operably linked to a Gal 10 promoter,

(iv) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is an immunogen operably linked to a Gal 10 promoter, and (b) inducing the Gal 10 promoter. [0012] In one embodiment, the method further comprises the steps of: (c) inducing the TEF1 promoter, and (d) providing gancyclovir to the yeast cell. In embodiments, the providing of gancyclovir to the yeast cell occurs in vitro. In embodiments, the providing of the gancyclovir to the yeast cell occurs in vivo , for example under conditions where the yeast cell is administered to a subject, gancyclovir can also be administered to the subject such that the providing of the gancyclovir to the yeast cell occurs in the subject.. In another embodiment, the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase. In another embodiment, the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

[0013] In another aspect the disclosure provides a recombinant microorganism for producing one or more heterologous proteins, the microorganism comprising a genetic construct for producing one or more heterologous proteins in the recombinant microorganism, wherein the construct comprises: (i) a nucleic acid comprising a removeable coding sequence flanked on either end by recombination sites oriented in the same direction, wherein said removeable coding sequence comprises at least one gene encoding a protein having a function essential to replication of the microorganism, and (ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to a regulatable promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the microorganism, thereby inhibiting cell replication, and (iii) at least one nucleic acid comprising a gene encoding a heterologous protein, wherein the gene encoding the heterologous protein is operably linked to a regulatable promoter.

[0014] In one embodiment, the regulatable promoter operably linked to the gene encoding the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein are inducible and activated by the same inducer. In another embodiment, the removeable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter, wherein the gene encoding a cytotoxic agent is downstream / 3’ to the gene encoding a protein having a function essential to replication of the microorganism. In another embodiment, the regulatable promoter operably linked to the cytotoxic agent is different from the regulatable promoter operably linked to the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein. In another embodiment, the cytotoxic agent is a herpes simplex virus (HSV) thymidine kinase. In another embodiment, the regulatable promoter is a TEF1 promoter. In another embodiment, the host microorganism is selected from the group consisting of a yeast, a bacterium, and a cyanobacterium. In another embodiment, the host microorganism is a yeast. In another embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the gene encoding the protein having a function essential to replication of the host microorganism is an endogenous DNA polymerase. In another embodiment, the heterologous recombinase enzyme is Cre recombinase and the recombination sites are lox sites. In another embodiment, the regulatable promoter is a Gal 10 promoter. In another embodiment, the heterologous protein is selected from the group consisting of a hormone, an anti-inflammatory protein, a cell-wall permeabilizing agent, an immunogen, a virus like particle (VLP) forming protein sequence, preferably wherein said VLP-forming protein sequence comprises a viral structural protein of functional fragment thereof, wherein said viral structural protein comprises a matrix protein, a capsid protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, or combinations thereof. In another embodiment, the cell wall permeablizing agent is selected from the group consisting of a cell wall degrading enzyme, and an inhibitor of cell wall biosynthesis. In another embodiment, the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase. In another embodiment, the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 Shows an exemplary nucleic acid comprising a removeable coding sequence flanked on either end by recombination sites. In particular, the nucleic acid comprises a lox71 site upstream of the regulatable TDH3 promoter that is is operably linked to the yeast polymerase 3 gene, which is essential for yeast replication, and a transcription termination signal. Downstream of the transcription termination signal is a regulatable TEF promoter operably linked to the Herpes simplex virus (HSV) thymidine kinase gene, followed by a transcription terminator and a lox 66 site.

[0016] FIG. 2 Shows a nucleic acid comprising a gene encoding a heterologous Cre recombinase enzyme. The gene encoding the heterologous recombinase enzyme is operably linked to a regulatable Gal 10 promoter. [0017] FIG. 3 Preliminary PCR data using the Cre and Amp primers indicated that all three transformants tested were integrated the construct into the genome.

DETAILED DESCRIPTION

Definitions

[0018] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0019] As used herein, “about” is understood by persons of ordinary skill in the art and may vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which the term “about” is used, “about” will mean up to plus or minus 10% of the particular term.

[0020] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0021] The phrase “a removable coding sequence flanked on either end by recombination sites oriented in the same direction” as used herein refers to a nucleic acid sequence that can be deleted by the action of a recombinase that is able to cause recombination between the recombination sites.

[0022] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a polymer of amino acid residues that is typically 12 or more amino acids in length.

Polypeptides less than 12 amino acids in length are referred to herein as “peptides.” The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “recombinant protein” refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide. In some exemplary embodiments, DNA or RNA encoding an expressed peptide, polypeptide or protein is inserted into the host chromosome via homologous recombination or other means well known in the art, and is so used to transform a host cell to produce the peptide or polypeptide. Similarly, the terms “recombinant polynucleotide” or “recombinant nucleic acid” or “recombinant DNA” are produced by recombinant techniques that are known to those of skill in the art (see e.g., Sambrook et ah, supra , Current Protocols in Molecular Biology, supra).

[0023] The term “therapeutic protein” as used herein refers broadly to protein-based products that can be used to promote well-being of humans or pets. A therapeutic protein can be a medicine, an antibody, an antigen, a vaccine, insulin, a nutritional supplement, etc. Therapeutic proteins can also be proteins that are an intermediate in the biosynthetic pathway of a protein that is ultimately used to promote well-being of humans or pets.

[0024] The term “host microorganism” as used herein, refers generally to a microscopic organism. Microorganisms can be prokaryotic or eukaryotic. Exemplary prokaryotic microorganisms include e.g. bacteria, fungi, archaea, cyanobacteria, etc. An exemplary fungi is the yeast, Saccharomyces cerevisiae. In exemplary embodiments, a “recombinant microorganism” is a microorganism that has been genetically altered and thereby expresses or encompasses a heterologous nucleic acid sequence and/or a heterologous protein. A “host microorganism” or equivalently a “host cell” is a cell used to produce products. As disclosed herein, a “host microorganism” is typically modified to express or overexpress selected genes, or to have attenuated expression of selected genes. Thus, a “host microorganism” or a “host cell” is a “recombinant host” or equivalently a “recombinant host cell.”

[0025] The expression “a function essential to replication of the microorganism” refer to those essential functions, typically carried out by proteins that allow a cell to replicate and divide. Exemplary proteins “essential to replication of the microorganism” include e.g., DNA polymerase proteins that are required for genome replication. The microorganism Saccharomyces cerevisiae , has at least six DNA polymerases (Sugino et al. (1995) Trends Biochem Sci 20(8):319-23).

[0026] The expression “recombination sites which are oriented in the same direction” as discussed in detail below, the orientation and location of asymmetric site specific recombination sites determines the type of recombination product that results from recombination between the sites. When acted upon by a recombinase, asymmetric recombination sites located on the same nucleic acid molecule and which are oriented in the same direction give rise to a deletion of the nucleic acid located between the recombination sites.

[0027] The term “endogenous” as used herein refers to a substance e.g., a nucleic acid, protein, etc. that is native to a microbial cell. Thus, an “endogenous” polynucleotide or polypeptide refers to a polynucleotide or polypeptide produced by the cell that is found inherently in that cell. In some exemplary embodiments an “endogenous” polypeptide or polynucleotide was present in the cell when the cell was originally isolated from nature i.e., the gene is “native to the cell.”

[0028] The term “heterologous” as used herein refers to a protein or nucleic acid which is in a non-native state. In the context of a cell and a protein or cell and a polynucleotide the term “heterologous” refers to a polypeptide or a polynucleotide that is not native to the cell in which it is expressed/produced. Thus, a polynucleotide or a polypeptide is “heterologous” to a cell when the polynucleotide and/or the polypeptide and the cell are not found in the same relationship to each other in nature. Therefore, a polynucleotide or polypeptide sequence is “heterologous” to an organism or a second sequence if it originates from a different organism, different cell type, or different species, or, if from the same species, it is modified from its original form. Thus, in an exemplary embodiment, a polynucleotide or polypeptide is “heterologous” when it is not naturally present in a given organism. For example, a polynucleotide sequence that is native to bacteriophage can be introduced into a host cell of Saccharomyces cerevisiae by recombinant methods, and the polynucleotide from bacteriophage is then heterologous to the S. cerevisiae cell. Thus, a “heterologous enzyme” is an enzyme that is not native to the cell in which it is expressed.

[0029] “Expression control sequences” or “regulatory sequences” are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, nucleotide sequences that affect RNA stability, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. In exemplary embodiments, “expression control sequences” interact specifically with cellular proteins involved in transcription (see e.g., Maniatis et al, Science , 236: 1237-1245 (1987); Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990)). In exemplary methods, an expression control sequence is operably linked to a polynucleotide sequence. [0030] The term “regulatable promoter” as used herein refers to a region of DNA that initiates transcription of a particular gene under specific conditions. The term includes inducible promoters and repressible promoters. Examples of inducible promoters include both positive inducible promoters, i.e., inducible promoters that are activated in the presence of the inducer, such as by interaction between the inducer and an activator molecule to enable binding of the combined entity to the inducible promoter to effect transcription of downstream genes controlled by the inducible promoter, and negative inducible promoters, i.e., inducible promoters that are activated in the presence of the inducer, such as by interaction between the inducer and a repressor to block or disable binding of the repressor to the inducible promoter, thereby removing suppression of transcription of downstream genes controlled by the inducible promoter. Examples of repressible promoters include both positive repressible promoters, i.e., promoters that are repressed in the presence of the repressor, such as by interaction between the repressor and an activator molecule to block or disable binding of the activator molecule to the repressible promoter, thereby removing activation of transcription of downstream genes controlled by the repressible promoter, and negative repressible promoters, i.e., promoters that are repressed in the presence of the repressor, such as by interaction between the repressor and a corepressor molecule to enable binding of the combined entity to the repressible promoter to effect transcription of downstream genes controlled by the repressible promoter. The term also includes promoters that can be regulated as both a positive inducible promoter and a negative inducible promoter, and promoters that respond to environmental queues, such as the presence or absence of light, the absence of a particular molecule, and any other promoter that can be specifically regulated by providing or removing a particular molecule or environmental queue.

[0031] The term “operably linked” as used herein refers to a polynucleotide sequence and an expression control sequence(s) that are functionally connected so as to permit expression of the polynucleotide sequence when the appropriate molecules (e.g, transcriptional activator proteins, inducer molecules, etc.) contact the expression control sequence(s). In exemplary embodiments, operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. In some exemplary embodiments, operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.

[0032] The expression “inhibiting cell replication” as used herein, refers to stopping or dramatically slowing cell growth and division.

[0033] The term “hormone” as used herein, refers broadly to signaling molecules produced by an organism that are and transported in the circulatory system to stimulate specific cells or tissues into action. In multicellular organisms hormones function in a multitude of ways to communicate between organs and tissues to regulate physiology and behavior. For example, hormones may influence activities such as digestion, metabolism, respiration, tissue function, sensory perception, sleep, excretion, lactation, stress induction, growth and development, movement, reproduction, and mood manipulation.

[0034] As used herein, the term “virus like particle” or “VLP” refers to a non-infectious nanostructures composed of viral structural proteins and lacking viral nucleic acid. A virus like particle morphologically resembles a virus, but, without more, lacks the ability to infect a host cell. VLPs are typically comprised of at least one structural component, such as a capsid or matrix protein that forms a particle shell.

[0035] As used herein, the term “enveloped virus like particle” or “eVLP” refers to a virus like particle that includes host cell-derived membranes, typically a lipid-based membrane obtained during the budding process as the vims emerges from the host cell. eVLPs typically comprise at least one matrix protein. In some cases, an eVLP may comprise 2 or 3, or more, different matrix proteins. In some cases, one or more of the matrix proteins are engineered to display an antigenic peptide sequence on the outer surface of the protein shell of the eVLP. For example, antigenic peptide sequence can be inserted into one or more matrix protein loop sequences such that the antigenic peptide sequence exposed on the outer protein surface of an assembled eVLP. In some cases, the eVLP is engineered to include one or more immunogenic peptides on the surface of the eVLP. In some cases, the eVLP component(s) are expressed in a host cell that further expresses one or more antigen (e.g., glycopeptides) that can embed in the lipid bilayer envelope of the eVLP.

[0036] As used herein, the term “non-enveloped vims like particle” refers to a VLP that does not include a host-cell derived membrane. The acronym neVLP refers to the term “non-enveloped vims like particle.” neVLPs can comprise at least one capsid protein. In some cases, an neVLP may comprise 2 or 3, or more, different capsid proteins. In some cases, one or more of the capsid proteins are engineered to display an antigenic peptide sequence on the outer surface of the neVLP. For example, antigenic peptide sequence can be inserted into one or more capsid protein loop sequences such that the antigenic peptide sequence is exposed on the outer protein surface of an assembled neVLP.

[0037] VLPs as described herein can be formed from one or more viral structural proteins or functional fragments thereof, for example viral stmctural proteins from SARS-CoV-2, Influenza, Respiratory syncytial virus (RSV), Simian Immunodeficiency Virus (SIV), Noravirus, and the like. In embodiments, nucleic acid sequences encoding VLP-forming protein sequences can include but are not limited to nucleic acid sequences encoding for one or more of a matrix protein, a capsid protein (e.g., nucleocapsid protein), a GAG protein, a GAG-homology protein, an envelope protein, fragments thereof, and combinations thereof. In exemplary embodiments, a VLP-forming protein sequence comprises a GAG protein (e.g., SIV GAG), or a GAG-homology protein or functional domain thereof, selected from the group consisting of Arc, ASPRV1, a Sushi-Class protein, a SCAN protein, or a PNMA protein. In additional or alternative embodiments, a VLP-forming protein sequence comprises a GAG-homology protein selected from the group consisting of PEG10, RTL3, RTL10, or RTL1. In additional or alternative exemplary embodiments, a VLP-forming protein sequence comprises yeast L-A GAG. In additional or alternative exemplary embodiments, a VLP-forming protein sequence comprises a matrix protein (e.g, Influenza Ml protein). In additional or alternative exemplary embodiments, a VLP-forming protein sequence comprises a capsid protein (e.g, a coronoviral N protein, Influenza NP). In additional or alternative exemplary embodiments, a VLP-forming protein sequence comprises an envelope protein (e.g., coronavirus E).

[0038] VLPs, including neVLPs and/or eVLPs can be engineered to include a nucleic acid binding peptide, which in turn can bind a specific nucleic acid binding site sequence. As described further below, one exemplary nucleic acid binding peptide is found in the MS2 coat protein, which binds an, e.g., 19-nucleotide, ribosomal binding site of the MS2 replicase mRNA, which folds into a hairpin loop structure. Typically one or more nucleic acid binding sites are included as a repeated array of nucleic acid binding sites to increase the amount of cognate protein localized to the nucleic acid. In some cases, the repeated sequence can compromise genetic stability of the recombinant coding sequence. In one embodiment, the nucleic acid binding sites in the repeated array are synonymous binding sites that are different in sequence and yet retain the cognate protein binding function. Such arrays of synonymous nucleic acid binding sites are described in, e.g., Wu et al., Genes Dev. 2015 Apr 15 (29(8); 876-886, as well as WO 2020/237100, and co-pending US Provisional Patent Application No. 63/118610, the contents of each of which are incorporated herein in their entirety for all purposes.

[0039] As used herein the term “toxin,” and “cytotoxin,” and “cytotoxic agent” are used interchangeably herein to refer to a substance or process which results in cell damage or cell death. A cytotoxic agent may also refer to an enzyme or other molecule that plays a part, or catalyzes a step in a cytotoxic process that ultimately leads to cell death. Examples include enzymes such as thymidine kinases which convert gancyclovir to a toxic product that kills cells.

[0040] As used herein, the term “common genetic control” a property of multiple nucleic acid sequences being regulated by the same promoter. The term includes nucleic acid arrangements in which the multiple nucleic acid sequences are positioned downstream of a single promoter that regulates the expression of both nucleic acid sequences. The term also includes nucleic acid arrangements in which one of the multiple nucleic acid sequences is positioned downstream of a first copy of the promoter and another of the multiple nucleic acid sequences is positioned downstream of a second copy of the promoter. It should be appreciated, that where multiple copies of a promoter are used in a scheme for expression of proteins under common genetic control, the copies need not be identical in sequence and minor variations in promoter sequence are tolerated so long as functional equivalency is maintained.

I. Introduction: Producing heterologous proteins in recombinant microorganisms

[0041] The present inventors have discovered a method for increasing cell metabolism during heterologous protein production while simultaneously preventing the host cell from replicating during and after heterologous protein production, thus reducing the risk of contaminating the environment with the genetically recombinant components. The present invention thus advantageously improves production of heterologous proteins in host microorganisms by reducing the metabolic burden of replication in the host, and freeing the resources normally used for replication to be diverted to production of the heterologous protein.

[0042] Accordingly, the disclosure provides compositions and methods for the production of heterologous proteins in a host cell (e.g., microorganism) utilizing a genetic construct that comprises: (i) a nucleic acid comprising a removable coding sequence flanked on either end by recombination sites oriented in the same direction wherein said removable coding sequence comprises at least one gene encoding a protein (e.g., DNA polymerase) having a function essential to replication of the host cell, and (ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to a regulatable promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the host cell, thereby inhibiting cell replication, and (iii) at least one nucleic acid comprising a gene encoding a heterologous protein of interest, wherein the gene encoding the heterologous protein is operably linked to a regulatable promoter. Deletion of the at least one gene encoding a protein having a function essential to replication of the host cell improves production of the heterologous protein by virtue of reducing demand for the cellular resources needed for cell replication. Furthermore, because the host cell cannot replicate after heterologous protein production, the cell dies, thereby reducing the risk of contaminating the environment with the genetically recombinant components. Finally, to ensure that any cells which fail to delete the removable coding sequence that comprises the at least one gene encoding a protein having a function essential to replication, in some embodiments, the removable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter which can be activated to kill those cells still capable of replication. Because the cytotoxic agent is located on the removable coding sequence, the cytotoxic agent is only produced by cells that fail to eliminate the removable coding sequence (see e g. FIG. 1).

[0043] As will be discussed in detail below, methods for producing heterologous proteins in a recombinant yeast include utilizing a genetic construct to inhibit cell replication during the induction phase. The inhibition of replication improves production of heterologous proteins by reducing the metabolic burden of replication and ensures genetically modified components do not escape into the environment.

[0044] In some cases, genome replication is inhibited by inhibiting expression or activity of an endogenous DNA polymerase. In some cases, DNA polymerase is inhibited by removing all or part of the genomic region encoding the endogenous DNA polymerase. In some cases, methods of producing heterologous proteins disclosed herein include inducing expression of a recombinant recombinase, such as CRE recombinase, and thereby inducing recombination between two lox sites ( e.g loxP sites) that are located in the genome and which flank at the genomic region encoding the endogenous DNA polymerase. In some embodiments, the CRE recombinase is under the genetic control of a regulatable promoter that is common to a nucleic acid sequence encoding a cell-wall permeabilizing agent and/or a nucleic acid sequence encoding an immunogen or a component thereof (e.g., an influenza hemagglutinin or neuraminidase, or a coronaviral spike protein), and/or a nucleic acid sequence encoding a VLP-forming protein sequence, such as a GAG protein (e.g., SIV or HIV GAG), GAG-homology protein (e.g., PEG10), a matrix protein (e.g., influenza M), a capsid protein (e.g., coronavirus N or influenza NP), an envelope protein (e.g., coronavirus E), fragments thereof, or combinations thereof. Thus, in some embodiments, yeast host cells described herein contain one or more recombination sites, such as loxP sites at or flanking a DNA polymerase encoding genomic region, and a nucleic acid encoding a heterologous recombinase, such as a CRE recombinase.

[0045] A recombination based approach for inhibiting cell replication can be particularly advantageous in forming a vaccine that is suitable for administration to a mammalian subject, wherein the vaccine contains, or is likely to contain, at least a portion of whole yeast cells because such cells will not replicate. For example, in some embodiments, VLPs described herein are induced with simultaneous or sequential recombination to inhibit replication, e.g., with simultaneous or sequential cell wall permeabilization, and cell culture supernatant containing VLPs are collected and used to form a vaccine. In some cases, cell culture supernatant used to form a vaccine further contains a yeast cell component, which can, e.g., provide an adjuvant effect. Additionally, or alternatively, in some embodiments described herein, the VLPs are from cell culture supernatant and non-replicable yeast cells are also harvested and admixed with formulation agents to produce the vaccine. Such an approach is not limited to vaccines per se, but can additionally or alternatively be used to administer, e.g., antibody(s), insulin, and the like as herein described. In embodiments, whole yeast cells and/or VLPs can comprise a therapeutic protein. In additional or alternative embodiments, whole yeast cells and/or VLPs can comprise an mRNA encoding a therapeutic protein, where the VLPs are engineered to include a nucleic acid binding peptide (e.g., MS2 peptide sequence), for binding to an MS2 ligand sequence included as part of the mRNA.

II. General Methods

[0046] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of recombinant genetics, immunology, and biochemistry. Basic texts disclosing the general terms in molecular biology and genetics include e.g., Lackie, Dictionary of Cell and Molecular Biology , Elsevier (5th ed. 2013). Basic texts disclosing methods in recombinant genetics and molecular biology include e.g., Sambrook et al, Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Press 4th Edition (Cold Spring Harbor, N.Y. 2012) and Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016). Basic texts disclosing the general methods and terms in biochemistry include e.g., Lehninger Principles of Biochemistry sixth edition, David L. Nelson and Michael M. Cox eds. W.H. Freeman (2012). Basic texts disclosing the general methods and terms immunology include Janeway's Immunobiology (Ninth Edition) by Kenneth M. Murphy and Casey Weaver (2017) Garland Science; Fundamental Immunology (Seventh Edition) by William E. Paul (2013) Lippincott, Williams and Wilkins.

Nucleic acid constructs

Cre-lox system

[0047] Cre-lox recombination is the site-specific recombination system from bacteriophage PI and is well known in the art (see e.g., Sternberg and Hamilton (1981) J. Mol Biol 25;150(4):467- 86). In the bacteriophage PI, the Cre-lox system functions to circularize the phage DNA into a plasmid on infection, separate interlinked plasmid rings so they are passed to both daughter bacteria equally and may help maintain phage copy numbers (Lobocka MB, et al. (2004). J. Bact. 186 (21): 7032-68).

[0048] The Cre-lox system has been developed as a tool for recombinant DNA manipulations such as e.g., to create deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. The Cre-lox system benefits from being highly versatile as it can be implemented both in eukaryotic and prokaryotic systems.

[0049] The Cre-lox system comprises the lox site specific recombination sites and the Cre recombinase which is a site specific recombinase that recognizes and acts on the lox sites. The lox sites are 34 bp in length and consisting of two 13-bp long palindromic repeats separated by an 8-bp long asymmetric core spacer sequence. Because the lox sites are asymmetric, the orientation and location of the lox sites (e.g., loxP sites) determines how the genetic material will be rearranged. In particular, if the lox sites are on the same DNA strand and are in opposite orientations, recombination results in an inversion of the DNA between the lox sites. If the lox sites are oriented in the same direction, the sequence between the lox sites is deleted as a circular DNA. Finally, if the sites are on separate DNA molecules, a translocation event is generated at the lox sites.

Regulatable promoters

[0050] Regulatable promoters are well known in the art (see e.g., Sambrook, supra). Regulatable promoters include both inducible promoters and repressible promoters. Examples of inducible promoters include e.g., both positive inducible promoters, i.e., inducible promoters that are activated in the presence of the inducer, such as by interaction between the inducer and an activator molecule to enable binding of the combined entity to the inducible promoter to effect transcription of downstream genes controlled by the inducible promoter, and negative inducible promoters, i.e., inducible promoters that are activated in the presence of the inducer, such as by interaction between the inducer and a repressor to block or disable binding of the repressor to the inducible promoter, thereby removing suppression of transcription of downstream genes controlled by the inducible promoter. Examples of repressible promoters include both positive repressible promoters, i.e., promoters that are repressed in the presence of the repressor, such as by interaction between the repressor and an activator molecule to block or disable binding of the activator molecule to the repressible promoter, thereby removing activation of transcription of downstream genes controlled by the repressible promoter, and negative repressible promoters, i.e., promoters that are repressed in the presence of the repressor, such as by interaction between the repressor and a corepressor molecule to enable binding of the combined entity to the repressible promoter to effect transcription of downstream genes controlled by the repressible promoter. The term also includes promoters that can be regulated as both a positive inducible promoter and a negative inducible promoter, and promoters that respond to environmental queues, such as the presence or absence of light, the absence of a particular molecule, and any other promoter that can be specifically regulated by providing or removing a particular molecule or environmental queue.

[0051] As another example, a positive repressible promoter can be used to regulate cell wall permeability by regulated repression of a cell wall biosynthesis pathway. For example, a recombinant yeast cell can be engineered to include a positive repressible promoter operably linked to a component of a cell wall biosynthesis pathway and to express an immunogen, e.g., in a regulated fashion. The recombinant yeast cell can be cultured under conditions to permit cell wall biosynthesis and then subsequently cell wall biosynthesis can be repressed by removal of the repressor. In some embodiments, the regulated repression of a cell wall biosynthesis pathway is provided by promoter replacement or insertion of a positive repressible promoter operably linked to an endogenous component of a cell wall biosynthesis pathway. Alternatively, an endogenous cell wall biosynthesis pathway component can be knocked out and an alternate, e.g., copy, introduced into the recombinant yeast cell that is operably linked to a positive repressible promoter.

[0052] As described herein, in some embodiments, the immunogen and cell-wall permeabilizing agent (e.g., cell wall degrading enzyme, cell wall biosynthesis toxin, etc.) are under the common genetic control of a regulatable promoter. Alternatively, in some embodiments, the immunogen and cell-wall permeabilizing agent are differentially regulated. In some embodiments, the regulated promoter is operably linked to the nucleic acid sequence encoding the cell wall permeabilizing agent. In some embodiments, a different, e.g., regulated, promoter is operably linked to the nucleic acid sequence encoding the immunogen or a component thereof.

[0053] In some cases, the promoter operably linked to the cell-wall permeabilizing agent is selected to induce or de-repress expression of the cell wall permeabilizing agent after the recombinant yeast have been cultured to a sufficient density (e.g., lxlO⁸ cells/mL, OD6oo> 10, or OD6OO 2 20 ) or growth phase (e.g., log phase, mid-log phase, or late-log phase growth). In some cases, the promoter operably linked to the cell-wall permeabilizing agent is selected to induce or de repress expression of the cell wall permeabilizing agent after the recombinant yeast have been harvested or after the recombinant yeast have been administered to a subject.

[0054] In some cases, the promoter operably linked to the immunogen or component thereof is selected to induce or de-repress expression of the immunogen prior to administration of the recombinant yeast to a subject. For example, immunogen production can be de-repressed or induced during culture of the recombinant yeast cells. In some methods of the present invention, immunogen expression is induced or de-repressed and then expression of the permeabilizing agent is induced or de-repressed. In some cases, the yield of expressed immunogen can be enhanced by inducing expression of cell wall permeabilizing agent after induction of immunogen expression. In other cases, e.g., where inefficient release of immunogen overwhelms the secretory capacity of the host cell, it may be preferable to induce expression of the cell wall permeabilizing agent prior to, or at the same time, as inducing the expression of the immunogen. As described herein, one exemplary method for simultaneous induction of both immunogen and cell wall permeabilization agent is to operably link the nucleic acid sequences encoding both the immunogen and the permeabilization agent to a regulatable common genetic control element.

[0055] In some embodiments, the nucleic acid sequence encoding the cell-wall permeabilizing agent is under control of a regulated promoter and the nucleic acid sequence encoding the immunogen is constitutively expressed.

[0056] A regulated promoter may comprise any suitable regulated promoter and a skilled artisan will be able to select a regulated promoter for a recombinant yeast cell according to a particular embodiment based on various considerations, including the nature of the wild-type yeast cell used in the production of the recombinant yeast cell, any desired type of control over the production of the immunogen and/or cell wall degrading enzyme, and any equipment and/or supplies needed to control expression of the VLP immunogen and cell wall degrading enzyme using a particular inducible promoter. Examples of suitable regulated promoters include inducible promoters, including positive inducible promoters, negative inducible promoters, and inducible promoters that can be regulated as both a positive inducible promoter and a negative inducible promoter, and repressible promoters, including positive repressible promoters, negative repressible promoters, and repressible promoters that can be regulated as both a positive repressible promoter and a negative repressible promoter. Examples of suitable regulated promoters include the Gal 10 inducible promoter, which activates transcription of genes controlled by the promoter in the presence of galactose, and the ADH2 promoter, which activates transcription in the absence of glucose. Other examples of regulated promoters include, but are not limited to, PTet, pTPl, pTEFl, pPYKl, pADHl, FMD1, pHXT7,pGALl, pGAL7, pGALlO, pPH05, pCUPl,and pDANl.

Heterologous proteins

[0057] In some embodiments, the heterologous protein is an immunogen that is or comprises a component of a virus like particle (VLP), such as a capsid protein, or a functional fragment thereof.

In some embodiments, the immunogen is or comprises a fusion protein comprising a first portion and a second portion, wherein the first portion comprises a capsid protein or functional fragment thereof and the second portion comprises an antigen. In some embodiments, the immunogen is or comprises a component of an enveloped VLP (eVLP), such as a matrix protein, or a functional fragment thereof. In some embodiments, the immunogen is or comprises a fusion protein comprising a first portion and a second portion, wherein the first portion comprises a matrix protein or functional fragment thereof and the second portion comprises an antigen. In some embodiments, the VLP comprises a fusion protein comprising a first portion and a second portion, wherein the first portion comprises a VLP-forming protein sequence (e.g., SIV or HIV-GAG,a capsid protein or a functional fragment thereof, GAG-homology protein or functional fragment thereof, matrix protein or a functional fragment thereof, envelope protein or a functional fragment thereof) and the second portion comprises an immunogen or a reporter polypeptide. In some cases, the reporter polypeptide is an enzyme. In some cases, the reporter polypeptide is a fluorescent protein. In embodiments, containing a reporter protein, such VLPs can be useful for tracking administration of VLPs to a subject and/or uptake of VLPs by cells of a subject. In some embodiments, the VLP comprises a fusion protein comprising a first portion and a second portion, wherein the first portion comprises a VLP-forming protein sequence (e.g., SIV or HIV-GAG,a capsid protein or a functional fragment thereof, GAG-homology protein or functional fragment thereof, matrix protein or a functional fragment thereof, envelope protein or a functional fragment thereof and the second portion comprises a nucleic acid binding peptide (e.g., MS2 peptide sequence) operable to bind, e.g., an mRNA that includes a ligand sequence (e.g., MS2 ligand sequence), where said mRNA encodes a therapeutic protein.

[0058] In some cases, the proteins from an influenza virus are selected from the group consisting of Ml matrix protein (e.g., human flu Ml matrix protein) or a functional fragment thereof, hemagluttinin or an immunogenic fragment thereof, and neuraminidase or an immunogenic fragment thereof. In some cases, the second nucleic acid encodes at least two proteins from an influenza virus selected from the group consisting of Ml matrix protein (e.g., human flu Ml matrix protein) or a functional fragment thereof, hemagluttinin or an immunogenic fragment thereof, and neuraminidase or an immunogenic fragment thereof. In some cases, the second nucleic acid encodes human flu Ml matrix protein, hemagglutinin, and neuraminidase.

[0059] In some cases, the proteins from a coronavirus are selected from the group consisting of a coronavirus spike protein (e.g., COVID-19 spike protein), or an immunogenic or functional fragment thereof, and a coronavirus Ml matrix protein (e.g., COVID-19 Ml matrix protein) or an immunogenic or functional fragment thereof. In some cases, the second nucleic acid encodes at least two proteins from a coronavirus selected from the group consisting of Ml matrix protein or an immunogenic or functional fragment thereof, and coronavirus spike protein, or an immunogenic or functional fragment thereof.

EXAMPLES

[0060] Example 1 : The following Example illustrates the nucleic acid sequence (SEQ ID NO: 1) corresponding to the nucleic acid construct shown in FIG. 1. The construct comprises a removeable coding sequence flanked on either end by recombination sites. The nucleic acid construct comprises a lox71 site upstream of the regulatable TDH3 promoter that is operably linked to the yeast polymerase 3 gene, which is essential for yeast replication, and a transcription termination signal. Downstream of the transcription termination signal is a regulatable TEF promoter operably linked to the Herpes simplex virus (HSV) thymidine kinase gene, followed by a transcription terminator and a lox 66 site. SEQ ID NO: 1

GAATTCTCTTCGTTCAACTTGTTTTCCTTGATGGCACGGTAGAGTCCACCATTTCCAT

CTGGTGATTGAGATAGGTTTACTGGGTCTTTCATTAGGAAATGCTTCCCGGTTAAAT

CAAAGGCAGGCAGGGTTCCCTGGTTGAAGAACGTAATTTGTTCTTTATTCAAGCCAA

AATAATTGTGTTCTTGAAAGTATGCCTCAGTAGCCGCTCTAGTGGGGCCTGATGTCA

TAATATACCAAGGAATTTCTACCTTTTTGTCCTTTACCATATCTTGCAACCTGATCAA

CTTTTCAGCTTGAATTTGAAAAAGAGATTTCTTAGAAGGCAATCCAATGTCGTAACA

GCCCTTTGGTTGAGAGGATCCTAACCGCGTACCTTGTCCGCCAGCCATTAAAATCAC

TGCGACTTCACCCTTGCCAATAGCTTCAAGGCCTAAACGCCAGTATTCATTTTCTTTC

TTACTGTTGCCAATAAGCGACTCGTACGAAGTAGGGGGCAATGGTGAAATTTCGAC

GCCAGTATCCTTAGAAGAGTTAGCTAGTGAGAATTTAATAGCATTTTGACAGTCTTC

CAGTAGTTTTGCAGGGGACCTCTTGGAAGATATTTGCTCCAGGTTTGAAAGCAATTC

TTCTTGGTCTTTGCGAGACAAGCTTTCCCAATTGTGGAAAAGTTGACTTTGTCCGGCT

TCAATGAATAGCTGTTTTGTGTCAGTCATAGTTGCAGTTCGATCTGGTGTTCTCCTTT

ATATCTGCGTTTCTTTGCAGCGTTCTTGCTTGACGGTTGATCTTGGTTTTGTGTGGTA

AAAGTATGCCTTAACGTAATAGGCAATTTTTCGTATTACGCGTTTAATTAAGATCTA

CCGTTCGTATAGCATACATTATACGAAGTTATCCTAGTTCATTATCAATACTCGCCAT

TTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAA

AAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTA

CACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACT

AAAT AT AAT GGAGCCCGCTTTTT AAGCTGGC ATCC AGAAAAAAAAAGAATCCC AGC

ACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAA

CTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTG

ATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCT

ATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGA

AAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATA

TAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATT

CTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAGAACTTAGTTTCGACCC

GCCGCCACCATGAGTGAAAAAAGATCCCTTCCCATGGTTGATGTGAAGATCGATGA

CGAGGAT ACTCCCC AGTT GGAAAAGA AAAT C AAACGGC AATC AAT AGATC AT GGTG

TTGGAAGTGAACCTGTTTCAACAATAGAGATTATTCCGAGTGATTCTTTTCGAAAAT AT AAT AGTC AAGGCTTC AAAGC AAAGGAT AC AGATTT AAT GGGT ACGC AATT AGAG

TCTACTTTTGAACAAGAGCTATCGCAAATGGAACATGATATGGCCGACCAAGAAGA

GCATGACCTGTCATCATTCGAGCGTAAGAAACTTCCAACCGATTTTGACCCAAGTTT

GTATGATATTTCTTTCCAACAAATTGATGCGGAACAGAGCGTACTGAATGGTATCAA

AGATGAAAATACATCTACCGTGGTAAGGTTTTTTGGTGTCACTAGTGAAGGACACTC

TGTACTTTGTAATGTTACAGGGTTCAAGAACTATCTTTACGTCCCAGCGCCCAATTCT

TCCGACGCTAACGATCAGGAGCAAATCAACAAGTTTGTGCACTATTTAAACGAAAC

ATTTGACCACGCTATTGATTCGATTGAAGTTGTATCTAAACAGTCTATCTGGGGTTAT

TCCGGAGATACCAAATTACCATTCTGGAAAATATACGTCACCTATCCGCATATGGTC

AACAAACTGCGTACTGCGTTTGAAAGAGGTCATCTTTCATTCAACTCGTGGTTTTCTA

ACGGCACGACTACTTATGATAACATTGCCTACACTTTAAGGTTAATGGTAGATTGTG

GAATTGTCGGTATGTCCTGGATAACATTACCAAAAGGAAAGTATTCGATGATTGAGC

CTAATAACAGAGTTTCCTCTTGTCAGTTGGAAGTTTCAATTAATTATCGTAACCTAAT

AGCACATCCTGCTGAGGGTGATTGGTCTCATACAGCTCCATTGCGTATCATGTCCTTT

GATATCGAGTGTGCTGGTAGGATTGGCGTCTTTCCGGAACCTGAATACGATCCCGTC

ATCCAAATTGCCAACGTTGTGAGTATTGCTGGCGCTAAGAAACCATTCATTCGTAAT

GTGTTTACTCTGAATACATGCTCACCCATAACAGGTTCAATGATTTTTTCCCACGCCA

CTGAAGAGGAAATGTTGAGCAATTGGCGTAACTTTATCATCAAAGTTGATCCTGATG

TTATCATTGGTTATAATACTACAAATTTTGATATCCCTTATCTTTTAAACCGTGCAAA

GGCGCTAAAGGTGAATGATTTCCCATATTTTGGAAGGTTAAAAACCGTTAAGCAAG

AAATT AAAGAGTCTGT GTTCTCTTCGAAGGCTT ATGGT AC AAGAGAAACC AAAAAT

GTCAATATTGACGGCCGATTACAGTTGGATCTTTTGCAATTTATTCAGCGTGAGTAT

AAACTAAGATCCTACACGTTGAATGCAGTCTCTGCGCACTTTTTAGGTGAACAGAAG

GAGGAT GT AC ATT AT AGC AT C ATTTCTGATCT AC A A A AT GGC GAT AGT GA A AC A AG

AAGAAGGTTGGCCGTTTACTGTTTGAAAGACGCCTACCTGCCTTTAAGGCTTATGGA

AAAACTAATGGCGTTAGTTAACTATACAGAAATGGCTCGTGTTACAGGTGTGCCATT

TTCATATTTACTAGCTCGTGGTCAACAAATTAAAGTTGTTTCTCAACTATTTCGAAAG

TGCCTGGAGATTGATACTGTGATACCTAACATGCAATCTCAGGCCTCTGATGACCAA

TATGAGGGTGCCACTGTTATTGAGCCTATTCGTGGTTATTACGATGTACCGATTGCA

ACTTTGGATTTCAATTCTTTATATCCAAGTATTATGATGGCGCACAACCTATGTTATA

CAACACTTTGTAACAAAGCTACTGTAGAGAGATTGAATCTTAAAATTGACGAAGACT ACGTCATAACACCTAATGGAGATTATTTTGTTACCACAAAAAGAAGGCGTGGTATAT

TACCAATTATTCTGGATGAATTAATAAGTGCTAGAAAACGCGCTAAAAAAGATCTG

AGAGATGAGAAGGATCCATTCAAAAGAGATGTTTTAAATGGTAGACAATTGGCTTT

GAAGATTTCAGCTAACTCTGTCTATGGTTTTACAGGAGCGACGGTGGGTAAATTGCC

ATGTTTAGCCATTTCTTCATCTGTTACTGCTTATGGTCGTACCATGATTTTAAAAACT

AAAACCGC AGTCC AAGAAAAAT ATT GT AT AAAGAAT GGTT AT AAGC ACGATGCCGT

TGTGGTTTACGGTGACACTGATTCCGTTATGGTAAAGTTTGGTACAACAGATTTAAA

GGAAGCTATGGATCTTGGTACCGAAGCTGCCAAATATGTCTCCACTCTATTCAAACA

TCCGATTAACTTAGAATTTGAAAAAGCATACTTCCCTTACCTTTTGATAAATAAAAA

GCGTTATGCAGGTTTATTCTGGACTAATCCTGACAAGTTTGACAAGTTGGACCAAAA

AGGCCTTGCTTCTGTCCGTCGTGATTCCTGTTCCTTGGTTTCTATTGTTATGAATAAA

GTTTTAAAGAAAATTTTAATTGAAAGAAATGTAGATGGTGCTTTAGCTTTTGTCAGA

GAAACTATCAATGATATTCTGCATAATAGAGTAGATATTTCAAAGTTGATTATATCA

AAGACGTTAGCCCCAAATTACACAAATCCACAGCCGCACGCCGTTTTGGCTGAACGT

ATGAAGAGGAGAGAGGGCGTTGGTCCAAATGTTGGTGATCGTGTGGACTATGTCAT

TATCGGTGGTAATGATAAACTTTACAATAGAGCAGAAGATCCATTATTTGTACTAGA

AAAC AAT ATT C AAGT GGATTCGCGCT ATT ATTT AACT AAT C AATT AC AAAATCC AAT

CATTAGTATTGTTGCACCTATTATTGGCGACAAACAGGCGAACGGTATGTTCGTTGT

GAAATCCATTAAAATTAACACAGGCTCTCAAAAAGGAGGCTTGATGAGCTTTATTAA

AAAAGTTGAGGCTTGTAAAAGTTGTAAAGGTCCGTTGAGGAAAGGTGAAGGCCCTC

TTTGTTCAAACTGTCTAGCAAGGTCTGGAGAATTATACATAAAGGCATTATACGATG

TCAGAGATTTAGAGGAAAAATACTCAAGATTATGGACACAATGCCAAAGGTGCGCT

GGTAACTTACATAGTGAAGTTTTGTGTTCAAATAAGAACTGTGACATTTTTTATATGC

GGGTT A AGGTT A A A A A AGAGC T GC AGGAGA A AGT AGA AC A ATT AAGC A A AT GGT A

ATAAAAAACGATAGGGTGGCACATCATATTAGGATTAAGAAAGGCTAACAACTTTT

T T AT G A AG AT G A AG AT G A A AT ATTT GGT GT GT C A A AT A A A AAGC T AGC T T GT GT GC T

TAAGTTTGTGTTTTTTTCTTGGCTTGTTGTGTTATGAATTTGTGGCTTTTTCTAATATT

AAATGAATGTAAGATCTCATTATAATGAATAAACAAATGTTTCTATAATCCATTGTG

AATGTTTTGTTGGATCTCTTCGCATATAACTACTGTATGTGCTATGGTATGGACTATG

GAATATGATTAAAGATAAGGAGCTCAGTTTAGACATGGAGGCCCAGAATACCCTCC

TTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTT AGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGG

CGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCC

CTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGG

TTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGA

TACAGTTCTCACATCACATCCGAACATAAACAACCCGGATTCTAGACCCGCCGCCAC

CATGGAGACTAGTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAGCAACCGAC

GTACGGCGTTGCGCCCTCGCCGGCAGCAAGAAGCCACGGAAGTCCGCCTGGAGCAG

AAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCTCACGGGATGGGGAAAAC

CACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGTCTACGTACC

CGAGCCGATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCT

ACACCACACAACACCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTG

GTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGACCGACGCCGT

TCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCT

CACCCTCATCTTCGACCGCCATCCCATCGCCGCCCTCCTGTGCTACCCGGCCGCGCG

ATACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCC

GCCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACA

TCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTG

GCCGCGATTCGCCGCGTTTACGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGC

GGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGACGGCCGTGCCGCCCCA

GGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGTTAT

TTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTACAACGTGT

TTGCCTGGGCCTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCACGTCTTTATCCT

GGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGG

GATGGTCCAGACCCACGTCACCACCCCCGGCTCCATACCGACGATCTGCGACCTGGC

GCGCACGTTTGCCCGGGAGATGGGGGAGGCTAACTAATAATCAGTACTGACAATAA

AAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTC

TATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCC

AG AT GC GA AGTT A AGT GC GC AGA A AGT A AT AT CAT GCGT C A AT C GT AT GT GA AT GC

TGGTCGCTATACTGCGCGACGCCTAGGCCTATAACTTCGTATAGCATACATTATACG

AACGGTAGTTTAAACGAGCTCGAATCATATATATGTATATATAAATAGATACTTGGA

AAAATCCAGATTCAAACAATGTTTTTGAAATAATGCTTCTCATGTTTAGAGGCAAGA TAATTCTGAGTATGTTTTGGGTATTTTATTGTCAGTAATTTGTAAACGCTGCAATTCT

AATGAGACCGAGCTTTGTTGCTGTATGTCATTGAACCAAGGGTGGTTCAAAGCTTCA

TCTATATTGTACCTTTCATCAGGATTTAGGACCAAAAGATTGGATATTAAATGTAGT

ACTGAGTCATCGATTTTATCCCAGTACGGAGAGTAAAACGCATACTTAGCTTGTAAG

ATCTGTTCCTTCAATGAAGGTGGCCCCAATTGATCACTGAATGGAGGAAAACCACAT

AAACAGACATATAATATGACACCAGCACTCCAAAGATCTACTTTTGATGTGTATCCT

TTCTTTGTGAGGACTTCGGGCGCTACATAAGAAGGCGTACCACAAAGTGTGTTTGTG

AATTGCATTTCTCCTGTAAATTTTGCTAGGCCAAAATCCGCTATTTTAACTTGAATAT

CAATTTCATCTTCATCCCATGGGCCAAGTTGGACTTGACTTGGATTTTCGCGCCTTGT

TATATTTAATAAGATATTTTCTGGCTTGATGTCTCTGTGAATGATGTTTTGCTCATGC

AGATACTTCAATCCGGTAAGTAACTGTTTGAATAGAGCTTTGGATTCATCCTGTCTC

AAACATGTTTTTCTGACGATTCTTTCAAATAGTTCGCCATCGTCGATCTTTTCCAGTA

CCAAATATTTTTGAATCTGAGATTTGCTGATTGGCTCTACAAAACTATCTAGTAAGTT

GACAATGTTTGGATGTTGCACTCTCATTAGGATGTTAGTTTCCTCCCTAAATTGTTTA

TTCTTTTTTTGGTCGTCATTTTGCTGAGCGTGGAATATTTTCGAATTC

[0061] Example 2: The following Example illustrates preparation of a nucleic acid sequence comprising a heterologous Cre recombinase enzyme, wherein the heterologous Cre recombinase is operably linked to a Gal 10 promoter.

Materials and Methods

[0062] Plasmids VB200809-1175ufz (pOCH) and VB200806-2136scs (pVS) were transformed into NEB DH5a cells and glycerol stocks were made. Plasmid DNA was isolated from pOCH transformants, and digested with Kpnl to linearize, leaving lOOObp of homologous sequence to the genomic OCH1 at both ends of the linearized DNA. The linearized construct is shown in FIG. 2. The sequence of the construct is provided below as SEQ ID NO: 10.

[0063] Cells were transformed using LiAc/ SS carrier DNA/ PEG, (Gietz, R.D., et al. (2007) Nature Protocols, Vol.2 No.l 31-35) and Electroporation. Once transformed into yeast strain BY4741, which are auxotrophic for methionine, the yeast integrate the linearized construct into the genome by homologous recombination with the OCH1 gene sequence located on the chromosome. [0064] Resulting transformants were plated on selective media, 2% YNB -Met, and grown at

30°C. After 14 days colonies were streaked for isolation on fresh 2% YNB -Met plates and again grown at 30°C. After sufficient growth (~5-7days) isolated colonies were used to inoculate 2% YNB -Met broth, and were grown shaking at 30°C. Cells were spun down at 500xg, supernatant was discarded, and pellets were resuspended in fresh media every 48 hours.

[0065] To confirm that the construct had integrated into the yeast genome, genomic DNA was isolated from three transformants, and that DNA was used as template for PCR. Four sets of primers have been designed to confirm integration, as well as rule out episomal expression are shown below.

[0066] Preliminary PCR data using the Cre and Amp primers indicated that all three transformants tested integrated the construct into the genome (FIG. 3).

Primer Sequences

Amp-F : TTATCCGCCTCCATCCAGTC (SEQ ID NO: 2)

Amp-R : CTGCGGCCAACTTACTTCTG (SEQ ID NO: 3)

Cre-F : CCAGCTTTGCCAGTTGATGCAAC (SEQ ID NO: 4)

Cre-R : GCTCTAGCCATATCTCTAGCGGCAC (SEQ ID NO: 5)

OCHMET-F : GGCTGGGCCTCAACTAAACG (SEQ ID NO: 6)

OCHMET-R : CGATGTTGTCACCAGTGTGTGCC (SEQ ID NO: 7)

OCHCRE-F : GT GC C GC T AG AG AT AT GGC T AG AGC (SEQ ID NO: 8)

OCHCRE-R : CTGCTGCAAGTGCGACAACC (SEQ ID NO: 9)

SEQ ID NO: 10

GGTACCCACCAGAGATTGGCACCCTTCCAGACATATTTCGTTCGCAAAGAACCATAA

AGATAAAGAACCCTATTCAACATACACCTACCACTCTCCAAGGCCAGGCGATGATTC

CACGCAAGAGGGTATTTTGTGGCCCGTACACTGTGTGAAAAACACCTGGGGTAGTA

ATTGGTTGACCAAATAATGGACCAAGTGGTCACTAAGCATATTAAGATTGTATACAA

GGGTTTCTTGACTGACCGTGAATACTACTCCGCCTTCCACGACATCTGGAACTTCCAT

A AGACC GAC AT GA AC A AGT AC TT AGA A A AGC AT CAT AC AG AC GAGGTTT AC ATTGT

CGGTGTAGCTTTGGAGTATTGTGTCAAAGCCACCGCCATTTCCGCTGCAGAACTAGG

TTATAAGACCACTGTCCTGCTGGATTACACAAGACCCATCAGCGATGATCCCGAAGT CAT C AAT AAGGTT A AGGAAGAGTTGA AGGCCC AC AAC ATC AATGTCGT GGAT AAAT

AAGAGCTGAATAATACTTCTTCAACCTGATGAACTAGGGTGGCTTGCAAATGCACAA

ATCTATATAACAATATCTATATATATGTATGTACACCGAATTCCGACATATGGAGAA

GGAATAATAAAATATTAACTAACGTCTTTACGCCTCTCTTTATTCTTTTTTGGGTAAA

TTGCTTAAACTATTTGGCCGGCCCACCGCGAAAAGATTTGGCTGGGCCTCAACTAAA

CGCGCCTTTTTGGACTTTTCACGTTGCAGGGACAGCAACGTCAAAACTTCTGCATTA

AGGTAGTTTGGTAGCTTGGTAGCCACTTTAGTATTTCTGCCTTCTTCGAATACCGACA

TTATTTCTCGCCAATCCACATTCTCTCTCCCCATCTGCATCCTTTTATATTTAATAGGG

ATAGGTTGTTTTAGTTCTTTGATTCCGTTTTCATTTCAAGAGCAATAATAGCAATTTG

GAAAAAGAAAGCAAGTAAAAGAAAGAAGAGATCTTATTTTTTGCTTTTTCTCTTGAG

GTCACATGATCGCAAAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTG

T GGT GGTT GGC AAAT GACT AATT AAGTT AGTC AAGGCGCC ATCCTC AT GAAAACTGT

GTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTC

GAT GAAA AAAAT AAGAT AT AT AT AAGGTT AAGTAAAGCGTCTGTT AG AAAGGAAGT

TTTTCCTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGT

CGTCAGATACATAGATACAATTCTATTACCCCCATCCATACACCCGCCGCCACCATG

CCATCTCATTTCGATACTGTTCAACTACACGCCGGCCAAGAGAACCCTGGTGACAAT

GCTCACAGATCCAGAGCTGTACCAATTTACGCCACCACTTCTTATGTTTTCGAAAAC

TCTAAGCATGGTTCGCAATTGTTTGGTCTAGAAGTTCCAGGTTACGTCTATTCCCGTT

TCCAAAACCCAACCAGTAATGTTTTGGAAGAAAGAATTGCTGCTTTAGAAGGTGGTG

CTGCTGCTTTGGCTGTTTCCTCCGGTCAAGCCGCTCAAACCCTTGCCATCCAAGGTTT

GGCACACACTGGTGACAACATCGTTTCCACTTCTTACTTATACGGTGGTACTTATAA

CCAGTTCAAAATCTCGTTCAAAAGATTTGGTATCGAGGCTAGATTTGTTGAAGGTGA

C AATCC AGAAGAATTCGAAAAGGTCTTTGAT GAAAGAACC AAGGCTGTTT ATTT GG

AAACCATTGGTAATCCAAAGTACAATGTTCCGGATTTTGAAAAAATTGTTGCAATTG

CTCACAAACACGGTATTCCAGTTGTCGTTGACAACACATTTGGTGCCGGTGGTTACT

TCTGTCAGCCAATTAAATACGGTGCTGATATTGTAACACATTCTGCTACCAAATGGA

TTGGTGGTCATGGTACTACTATCGGTGGTATTATTGTTGACTCTGGTAAGTTCCCATG

GAAGGACTACCCAGAAAAGTTCCCTCAATTCTCTCAACCTGCCGAAGGATATCACG

GTACTATCTACAATGAAGCCTACGGTAACTTGGCATACATCGTTCATGTTAGAACTG

AACTATTAAGAGATTTGGGTCCATTGATGAACCCATTTGCCTCTTTCTTGCTACTACA AGGTGTTGAAACATTATCTTTGAGAGCTGAAAGACACGGTGAAAATGCATTGAAGT

TAGCCAAATGGTTAGAACAATCCCCATACGTATCTTGGGTTTCATACCCTGGTTTAG

CATCTCATTCTCATCATGAAAATGCTAAGAAGTATCTATCTAACGGTTTCGGTGGTG

TCTTATCTTTCGGTGTAAAAGACTTACCAAATGCCGACAAGGAAACTGACCCATTCA

AACTTTCTGGTGCTCAAGTTGTTGACAATTTAAAGCTTGCCTCTAACTTGGCCAATGT

TGGTGATGCCAAGACCTTAGTCATTGCTCCATACTTCACTACCCACAAACAATTAAA

TGACAAAGAAAAGTTGGCATCTGGTGTTACCAAGGACTTAATTCGTGTCTCTGTTGG

TATCGAATTTATTGATGACATTATTGCAGACTTCCAGCAATCTTTTGAAACTGTTTTC

GCTGGCCAAAAACCATAATAAGTGTGCGTAATGAGTTGTAAAATTATGTATAAACCT

CCATATCCAACTTCCAATTTAATCTTTCTTTTTTAATTTTCACTTATTTGCGATACAGA

AAGAGGATCCAACGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGA

CCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGC

TCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGA

AAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCAC

A AC AT AT A AGT A AG ATT AG AT AT GG AT AT GT AT AT GG AT AT GT AT AT GGT GGT A AT G

CCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTT

TTGGTCGACCCCGCCGCCACCATGTCTAATTTGTTGACTGTTCATCAAAATTTGCCAG

CTTTGCCAGTTGATGCAACGTCTGATGAGGTAAGAAAGAATTTGATGGATATGTTTA

GAGATAGACAAGCATTCTCTGAACATACTTGGAAAATGTTGTTGTCTGTTTGTAGAT

CTTGGGCTGCTTGGTGTAAATTAAATAATAGAAAATGGTTTCCAGCTGAACCAGAAG

ATGTTAGAGATTATTTGTTGTATTTGCAAGCTAGAGGTTTGGCTGTTAAAACTATAC

AACAACATTTGGGTCAATTAAATATGTTGCATAGGAGATCTGGTTTGCCTAGACCAT

CTGATTCTAATGCTGTATCTTTGGTTATGAGAAGGATTAGAAAAGAAAATGTTGATG

CAGGTGAAAGAGCTAAACAAGCATTGGCGTTTGAAAGAACTGATTTTGATCAAGTT

AGATCTTTGATGGAAAATTCTGATAGATGTCAAGATATAAGAAACTTGGCTTTCTTG

GGAATT GCTT AT AAT AC ATT GTT GAGAATTGCTGAAATT GCT AGA ATT AGAGTT AAA

GAC ATTTCT AGAACTGAT GGTGGT AGAAT GTT GATT CAT ATTGGT AGAACT AAAAC A

TTGGTTTCAACTGCTGGCGTTGAGAAAGCATTGTCTTTGGGTGTTACTAAATTGGTTG

AAAGATGGATTTCTGTATCTGGTGTGGCTGATGACCCAAATAATTATTTATTTTGTAG

AGTTAGAAAGAATGGTGTTGCGGCTCCATCTGCTACATCTCAGCTCTCTACCCGTGC

CTTGGAAGGAATTTTTGAAGCAACTCATAGATTGATTTATGGTGCTAAAGATGATTC TGGTCAAAGATATTTAGCATGGTCTGGTCATTCTGCTAGAGTTGGTGCCGCTAGAGA

TATGGCTAGAGCTGGGGTATCTATTCCTGAAATTATGCAAGCTGGTGGTTGGACTAA

TGTTAACATCGTCATGAACTATATTAGAAATTTGGATTCTGAAACTGGTGCAATGGT

T AG AC TC TT GGA AG AT GGT GATT A AT A AGAC CGCGT CAT GT A ATT AGTT AT GTC ACG

CTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACA

ACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTA

TTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTAT

ACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCGAATT

CCGAGGCTCTCCTTTCATCATACCCTCTCTAAATAAAATTTATTCCTAGTTATTCCTTT

CTTCTTCATATCCTTAATCATGCATTCACTGCCATGTGCAAATAAAGGCTCTTGTGGT

AGAAACTTTTAGCTCAAACGTGGTAAAACTATCTCAACGTCCTTCCGTAGACAACTG

GTGAGCAGTGCTTCTGTAACACTGCAGATCTCCAATGATAAATCGATACCGTACTAA

CTGCCCGCACTATAATGAACTCTTTGTATCCGTAACTATTTAAAATATTCATTCATTT

CTATAAGCTATATGTTTATATTTAGATTAGAGGGGTTAAAAGAAAGTTTTTCTCGAA

AGCTTAATTTTAGCTTTTACTTTGAACTTCTAGTAATTGCGAGGCAGTATCGACAATG

CAATCTCTGAACTTATTAAATTGGAATCCTAATAAATTTTTGGTTTTGCGGTTGTCGC

ACTTGCAGCAGTTTTTTGTCAAAAAGGTTGAGCCGCTACCAGGTTCGCCAGTTGCTA

TCTTGCCTTTTAACTGTGGAAATTCCTCATTCAAGATATCCAGCGCTTCTTGAGAGCA

AAACATATCTTCACATAAGAATAGTCTTTGGCCAGCGCATTCGGGTTTCTCAAATGC

AAGTAAATGAGCTTTTGAAACATCGCGAACGTCAATAAAAGGACCACTGTAATTAT

AAAAATT GTCGCCT AATTT AT AACT AACC AAATTGGC AAT AATGGCTGAAGAGCT AT

TTATTCCATTTCTAAGAGAGTCGGCAAATAGCTGAGGGCCAAAAACAAATCCTGGGT

TGATGGTTGATAGCGTAAATTTGATGCTTGATTGGTTTTCCTCGAGAAAATCCCAAG

CAGTTTTTTCAGCAAATTTCTTGGAACCACAGTATGCGGAAACCGCGTTAGCTTGAC

AACTTTCCCAAGTATCTTTGTTCCAACTTTCCTCATTGACAACGAAACTAGTGTCCTT

CATATCTCCGGGAGATGCAAGGGCAGGTACC

[0067] Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description. It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims.

Claims

What is claimed is:

1. A genetic construct for producing one or more heterologous proteins in a host microorganism, the construct comprising:

(i) a nucleic acid comprising a removable coding sequence flanked on either end by recombination sites oriented in the same direction wherein said removable coding sequence comprises at least one gene encoding a protein having a function essential to replication of the microorganism, and

2. The genetic construct of claim 1, wherein the regulatable promoter operably linked to the gene encoding the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein are inducible and activated by the same inducer.

3. The genetic construct of claim 1, wherein the removable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter, wherein the gene encoding a cytotoxic agent is downstream (3’) to the gene encoding a protein having a function essential to replication of the microorganism.

4. The genetic construct of claim 3, wherein the regulatable promoter operably linked to the cytotoxic agent is different from the regulatable promoter operably linked to the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein.

5. The genetic construct of claim 3 or claim 4, wherein the cytotoxic agent is a herpes simplex virus (HSV) thymidine kinase.

6. The genetic construct of any one of claims 3-5, wherein the regulatable promoter operably linked to the cytotoxic agent is a TEF1 promoter.

7. The genetic construct of any one of claims 1-6, wherein the host microorganism is selected from the group consisting of a yeast, a bacterium, and a cyanobacterium.

8. The genetic construct of claim 7, wherein the host microorganism is a yeast.

9. The genetic construct of claim 8, wherein the yeast is Saccharomyces cerevisiae.

10. The genetic construct of any one of claims 1-9, wherein the gene encoding the protein having a function essential to replication of the host microorganism is an endogenous DNA polymerase.

11. The genetic construct of claim any one of claims 1-10, wherein the heterologous recombinase enzyme is Cre recombinase and the recombination sites are lox sites.

12. The genetic construct of claim 1 or 2, wherein the regulatable promoter is a GallO promoter.

13. The genetic construct of any one of claims 1-12, wherein the heterologous protein is selected from the group consisting of a hormone, an anti-inflammatory protein, a cell- wall permeabilizing agent, an immunogen, a virus like particle (VLP) forming protein sequence, preferably wherein said VLP -forming protein sequence comprises a viral structural protein or functional fragment thereof, and combinations thereof.

14. The genetic construct of claim 13, wherein said viral structural protein comprises a capsid protein, a matrix protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, and combinations thereof.

15. The genetic construct of claim 13, wherein the cell wall permeablizing agent is selected from the group consisting of a cell wall degrading enzyme, and an inhibitor of cell wall biosynthesis.

16. The genetic construct of claim 15, wherein the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase.

17. The genetic construct of claim 13, wherein the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

18. A genetic construct for producing one or more heterologous proteins in a yeast cell, comprising:

(i) a nucleic acid comprising a removable coding sequence flanked on either end by recombination sites oriented in the same direction wherein said removable coding sequence comprises an endogenous DNA polymerase, and a gene encoding a herpes simplex virus (HSV) thymidine kinase operably linked to a TEF1 promoter, wherein the gene encoding the HSV thymidine kinase is located adjacent to and downstream of the endogenous DNA polymerase,

(ii) a nucleic acid comprising a heterologous Cre recombinase enzyme, wherein the heterologous Cre recombinase is operably linked to a Gal 10 promoter, and wherein the Cre recombinase is capable of acting on the lox recombination sites to recombine the lox recombination sites and delete the gene encoding the a endogenous DNA polymerase, thereby inhibiting cell replication,

(iii) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is a cell wall degrading enzyme operably linked to a Gal 10 promoter, and

(iv) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is an immunogen operably linked to a Gal 10 promoter.

19. The genetic construct of claim 18, wherein the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase.

20. The genetic construct of claim 18, wherein the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

21. A method for producing a heterologous protein, the method comprising:

(a) cultivating a host microorganism that comprises:

(i) a nucleic acid comprising a removable coding sequence flanked on either end by recombination sites oriented in the same direction, wherein said removable coding sequence comprises at least one gene encoding a protein having a function essential to replication of the microorganism, and

(ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to an inducible promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the microorganism, thereby inhibiting cell replication, and

(iii) at least one nucleic acid comprising a gene encoding a heterologous protein, wherein the gene encoding the heterologous protein is operably linked to an inducible promoter, and

(b) inducing the inducible promoter.

22. The method of claim 21, wherein the inducible promoter operably linked to the gene encoding the heterologous recombinase enzyme and the inducible promoter operably linked to the gene encoding the heterologous protein are activated by the same inducer.

23. The method of claim 21 or claim 22, wherein the removable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter, wherein the gene encoding a cytotoxic agent is downstream / 3’ to the gene encoding a protein having a function essential to replication of the microorganism.

24. The genetic construct of claim 23, wherein the regulatable promoter operably linked to the cytotoxic agent is different from the regulatable promoter operably linked to the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein.

25. The method of claim 23 or claim 24, further comprising the step of inducing the regulatable promoter operably linked to the cytotoxic agent, thereby inducing expression of the cytotoxic agent.

26. The method of any one of claims 23-25, wherein the cytotoxic agent acts on a precursor compound to convert the precursor compound to a toxic product.

27. The method of claim 26, further comprising providing the precursor compound to the host microorganism.

28. The method of claim 26 or claim 27, wherein the cytotoxic agent is a herpes simplex virus (HSV) thymidine kinase, the regulatable promoter is a TEF1 promoter, and the precursor compound is gancyclovir.

29. The method of any one of claims 21-28, wherein the host microorganism is selected from the group consisting of a yeast, a bacterium, and a cyanobacterium.

30. The method of claim 29, wherein the host microorganism is a yeast.

31. The method of claim 30, wherein the yeast is Saccharomyces cerevisiae.

32. The method of any one of claims 21-31, wherein the gene encoding the protein having a function essential to replication of the host microorganism is an endogenous DNA polymerase.

33. The method of any one of claims 21-32, wherein the heterologous recombinase enzyme is Cre recombinase and the recombination sites are lox sites.

34. The method of claim 21 or 22, wherein the inducible promoter is a Gal 10 promoter.

35. The method of any one of claims 21-34, wherein the heterologous protein is selected from the group consisting of a hormone, an anti-inflammatory protein, a cell-wall permeabilizing agent, an immunogen, a virus like particle (VLP) forming protein sequence, preferably wherein said VLP -forming protein sequence comprises a viral structural protein or functional fragment thereof, and combinations thereof.

36. The method of claim 35, wherein said viral structural protein comprises a capsid protein, a matrix protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, and combinations thereof.

37. The method of claim 35 or claim 36, wherein the cell wall permeablizing agent is selected from the group consisting of a cell wall degrading enzyme, and an inhibitor of cell wall biosynthesis.

38. The method of claim 37, wherein the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase.

39. The method of any one of claims 35-38, wherein the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

40. A method for producing a vaccine, the method comprising:

(a) cultivating a yeast cell, that comprises:

(i) a nucleic acid comprising a removable coding sequence flanked on either end by recombination sites oriented in the same direction, wherein said removable coding sequence comprises an endogenous DNA polymerase, and a gene encoding a herpes simplex virus (HSV) thymidine kinase operably linked to a TEF1 promoter, wherein the gene encoding the HSV thymidine kinase is located adjacent to and downstream of the endogenous DNA polymerase, and

(ii) a nucleic acid comprising a gene encoding a heterologous recombinase enzyme, wherein the gene encoding the heterologous recombinase enzyme is operably linked to an Gal 10 promoter, and wherein the heterologous recombinase is capable of acting on the recombination sites to recombine the recombination sites and delete the gene encoding the protein having a function essential to replication of the microorganism, thereby inhibiting cell replication,

(iii) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is a cell wall degrading enzyme operably linked to a Gal 10 promoter,

(iv) a nucleic acid encoding a heterologous gene encoding a heterologous protein that is an immunogen operably linked to a Gal 10 promoter, and

(b) inducing the Gal 10 promoter.

41. The method of claim 40, further comprising the steps of:

(c) inducing the TEF1 promoter, and

(d) providing gancyclovir to the yeast cell.

42. The method of claim 40 or claim 41, wherein the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase.

43. The method of any one of claims 40-42, wherein the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.

44. The method of any one of claims 40-43, wherein the yeast cell further comprises a nucleic acid encoding a heterologous gene encoding a heterologous protein that is a VLP- forming protein sequence, preferably wherein said VLP-forming protein sequence comprises a viral structural protein or functional fragment thereof.

45. The method of claim 44, wherein said viral structural protein comprises a capsid protein, a matrix protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, or combinations thereof.

46. A recombinant microorganism for producing one or more heterologous proteins, the microorganism comprising a genetic construct for producing one or more heterologous proteins in the recombinant microorganism, wherein the construct comprises:

47. The recombinant microorganism of claim 46, wherein the regulatable promoter operably linked to the gene encoding the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein are inducible and activated by the same inducer.

48. The recombinant microorganism of claim 46 or claim 47, wherein the removable coding sequence further comprises a gene encoding a cytotoxic agent operably linked to a regulatable promoter, wherein the gene encoding a cytotoxic agent is downstream / 3’ to the gene encoding a protein having a function essential to replication of the microorganism.

49. The recombinant microorganism of claim 48, wherein the regulatable promoter operably linked to the cytotoxic agent is different from the regulatable promoter operably linked to the heterologous recombinase enzyme and the regulatable promoter operably linked to the gene encoding the heterologous protein.

50. The recombinant microorganism of claim 48 or claim 49, wherein the cytotoxic agent is a herpes simplex virus (HSV) thymidine kinase.

51. The recombinant microorganism of any one of claims 48-50, wherein the regulatable promoter operably linked to the cytotoxic agent is a TEF1 promoter.

52. The recombinant microorganism of any one of claims 46-51, wherein the host microorganism is selected from the group consisting of a yeast, a bacterium, and a cyanobacterium.

53. The recombinant microorganism of claim 52, wherein the host microorganism is a yeast.

54. The recombinant microorganism of claim 53, wherein the yeast is Saccharomyces cerevisiae.

55. The recombinant microorganism of any one of claims 46-54, wherein the gene encoding the protein having a function essential to replication of the host microorganism is an endogenous DNA polymerase.

56. The recombinant microorganism of any one of claims 46-55, wherein the heterologous recombinase enzyme is Cre recombinase and the recombination sites are lox sites.

57. The recombinant microorganism of claim 46 or 47, wherein the regulatable promoter is a Gal 10 promoter.

58. The recombinant microorganism of any one of claims 46-57, wherein the heterologous protein is selected from the group consisting of a hormone, an anti-inflammatory protein, a cell-wall permeabilizing agent, an immunogen, a virus like particle (VLP) forming protein sequence, preferably wherein said VLP-forming protein sequence comprises a viral structural protein or functional fragment thereof, and combinations thereof.

59. The recombinant microorganism of claim 58, wherein said viral structural protein comprises a capsid protein, a matrix protein, a GAG protein, a GAG-homology protein, an envelope protein, functional fragments thereof, or combinations thereof.

60. The recombinant microorganism of claim 58 or claim 59, wherein the cell wall permeablizing agent is selected from the group consisting of a cell wall degrading enzyme, and an inhibitor of cell wall biosynthesis.

61. The recombinant microorganism of claim 60, wherein the cell wall degrading enzyme is selected from the group consisting of a mannase, a glucanase, and a chitinase.

62. The recombinant microorganism of any one of claims 57-61, wherein the immunogen is selected from the group consisting of an influenza hemagglutinin, an influenza neuraminidase, and a coronaviral spike protein.