Patent Application
20240327881
Terpenes are a large class of hydrocarbons that are produced in many organisms. They are derived by linking units of isoprene (C5H8), and are classified by the number of isoprene units present. Hemiterpenes consist of a single isoprene unit. Isoprene itself is considered the only hemiterpene. Monoterpenes are made of two isoprene units, and have the molecular formula C10H16. Examples of monoterpenes are geraniol, limonene, and terpineol. Sesquiterpenes are composed of three isoprene units, and have the molecular formula C15H24. Examples of sesquiterpenes are farnesene, farnesol and patchoulol. Diterpenes are made of four isoprene units, and have the molecular formula C20H32. Examples of diterpenes are cafestol, kahweol, cembrene, and taxadiene. Sesterterpenes are made of five isoprene units, and have the molecular formula C25H40. An example of a sesterterpene is geranylfarnesol. Triterpenes consist of six isoprene units, and have the molecular formula C30H48. Tetraterpenes contain eight isoprene units, and have the molecular formula C40H64. Biologically important tetraterpenes include the acyclic lycopene, the monocyclic gamma-carotene, and the bicyclic alpha- and beta-carotenes. Polyterpenes consist of long chains of many isoprene units. Natural rubber consists of polyisoprene in which the double bonds are in the cis conformation.
When terpenes are chemically modified (e.g., via oxidation or rearrangement of the carbon skeleton) the resulting compounds are generally referred to as terpenoids, which are also known as isoprenoids. Isoprenoids play many important biological roles, for example, as quinones in electron transport chains, as components of membranes, in subcellular targeting and regulation via protein prenylation, as photosynthetic pigments including carotenoids and chlorophyll, as hormones and cofactors, and as plant defense compounds. They are industrially useful as antibiotics, hormones, anticancer drugs, insecticides, and chemicals.
Terpenes are biosynthesized through condensations of isopentenyl pyrophosphate (isopentenyl diphosphate or IPP) and its isomer dimethylallyl pyrophosphate (dimethylallyl diphosphate or DMAPP). Two pathways are known to generate IPP and DMAPP, namely the mevalonate-dependent (MEV) pathway of eukaryotes, and the mevalonate-independent or deoxyxylulose-5-phosphate (DXP) pathway of prokaryotes. Plants use both the MEV pathway and the DXP pathway. IPP and DMAPP in turn are condensed to polyprenyl diphosphates (e.g., geranyl disphosphate or GPP, farnesyl diphosphate or FPP, and geranylgeranyl diphosphate or GGPP) through the action of prenyl disphosphate synthases (e.g., GPP synthase, FPP synthase, and GGPP synthase, respectively).
Traditionally, isoprenoids have been manufactured by extraction from natural sources such as plants, microbes, and animals. However, the yield by way of extraction is usually very low due to a number of profound limitations. First, most isoprenoids accumulate in nature in only small amounts. Second, the source organisms in general are not amenable to the large-scale cultivation that is necessary to produce commercially viable quantities of a desired isoprenoid. Third, the requirement of certain toxic solvents for isoprenoid extraction necessitates special handling and disposal procedures, thus complicating the commercial production of isoprenoids.
The elucidation of the MEV and DXP metabolic pathways has made biosynthetic production of isoprenoids feasible. For instance, microbes have been engineered to overexpress a part of or the entire mevalonate pathway for production of an isoprenoid named amorpha-4,11-diene. Other efforts have focused on balancing the pool of glyceraldehyde-3-phosphate and pyruvate, or on increasing the expression of 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and IPP isomerase (idi).
Nevertheless, given the very large quantities of isoprenoid products needed for many commercial applications, there remains a need for expression systems and fermentation procedures that produce even more isoprenoids than available with current technologies. Optimal redirection of microbial metabolism toward isoprenoid production requires that the introduced biosynthetic pathway is properly engineered both to funnel carbon to isoprenoid production efficiently and to prevent buildup of toxic levels of metabolic intermediates over a sustained period of time. Provided herein are compositions and methods that address this need and provide related advantages as well.
Provided herein are compositions and methods for producing one or more isoprenoid compounds, such as gamma-ambryl acetate (GAA), in a host cell, such as a yeast cell, that is genetically modified to express the enzymes of an isoprenoid biosynthetic pathway, such as a pathway for making GAA. Using the compositions and methods of the present invention, the host cell may be genetically modified to express one or more enzymes of an isoprenoid biosynthetic pathway, such as an enzyme capable of converting manooloxy to GAA. The host cell may then be cultured in a medium, for example, in the presence of an agent that regulates expression of the one or more enzymes. The host cell may further be incubated for a time sufficient to allow for production of an isoprenoid compound by the host cell. The isoprenoid compound may then be separated from the host cell or from the medium.
In one aspect, the invention provides for a genetically modified host cell capable of producing gamma-ambryl acetate (GAA), wherein the genetically modified host cell contains one or more heterologous nucleic acids that each, independently, encodes an enzyme having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12. In an embodiment, the enzyme has the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12.
In another aspect, the invention provides for a genetically modified host cell capable of producing gamma-ambryl acetate (GAA), wherein the genetically modified host cell contains one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting manooloxy to GAA. In an embodiment, the enzyme capable of converting manooloxy to GAA is a Baeyer-Villiger monooxygenase (BVMO).
In a further embodiment, the genetically modified host cell further contains one or more heterologous nucleic acids that each, independently, encodes one or more enzymes of a pathway for making GAA. In a further embodiment, the genetically modified host cell further contains one or more enzymes having the amino acid sequence of SEQ ID NO. 18, 21, 24, 27, 41, 44, 47, or 48. In a further embodiment, the genetically modified host cell further contains one or more of (a) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting one or more IPP, DMAPP, GPP, FPP, or GGPP into GPP, FPP, GGPP, or CPP; (b) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting CPP to E-copalol; (c) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting E-copalol to E-copalal; or (d) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting E-copalal to manooloxy. In a further embodiment, the genetically modified host cell further contains one or more of a CPP synthase, an Erg20, a GPP synthase, a GGPP synthase, a CPP pyrophosphatase, an alcohol dehydrogenase, or an enal-cleaving enzyme.
In an embodiment, expression of one or more of the enzymes provided by the invention is under the control of a single transcriptional regulator. In a further embodiment, expression of one or more of the enzymes provided by the invention is under the control of multiple transcriptional regulators.
In an embodiment, the genetically modified host cell is a yeast cell or a yeast strain. In a further embodiment, the yeast cell or the yeast strain is Saccharomyces cerevisiae.
In another aspect, the invention provides for a fermentation composition containing the genetically modified host cell disclosed herein, optionally an overlay, and GAA produced by the genetically modified host cell.
In another aspect, the invention provides for a method of producing GAA involving culturing the genetically modified host cell disclosed herein in a medium with a carbon source under conditions suitable for making GAA, optionally providing an overlay, and recovering GAA from the genetically modified host cell, the overlay, or the medium.
In another aspect, the invention provides for a non-naturally occurring enzyme capable of converting manooloxy to GAA having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12. In an embodiment, the non-naturally occurring enzyme comprises the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12.
As used herein, the singular forms “a,” “an,” and, “the” include plural reference unless the context clearly dictates otherwise.
As used herein, the term “about” when modifying a numerical value or range herein includes normal variation encountered in the field, and includes plus or minus 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) of the numerical value or end points of the numerical range. Thus, a value of 10 includes all numerical values from 9 to 11. All numerical ranges described herein include the endpoints of the range unless otherwise noted, and all numerical values in-between the end points, to the first significant digit.
As used herein, the term “capable of producing” refers to a host cell which is genetically modified to include the enzymes necessary for the production of a given compound in accordance with a biochemical pathway that produces the compound. For example, a cell (e.g., a yeast cell) “capable of producing” an isoprenoid compound is one that contains the enzymes necessary for production of the isoprenoid compound according to the isoprenoid biosynthetic pathway.
As used herein, the term “exogenous” refers a substance or compound that originated outside an organism or cell. The exogenous substance or compound can retain its normal function or activity when introduced into an organism or host cell described herein.
As used herein, the term “fermentation composition” refers to a composition which contains genetically modified host cells and products or metabolites produced by the genetically modified host cells. An example of a fermentation composition is a whole cell broth, which may be the entire contents of a vessel, including cells, aqueous phase, and compounds produced from the genetically modified host cells.
As used herein, the term “gene” refers to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, gRNA, or micro RNA.
A “genetic pathway” or “biosynthetic pathway” as used herein refer to a set of at least two different coding sequences, where the coding sequences encode enzymes that catalyze different parts of a synthetic pathway to form a desired product (e.g., an isoprenoid). In a genetic pathway a first encoded enzyme uses a substrate to make a first product which in turn is used as a substrate for a second encoded enzyme to make a second product. In some embodiments, the genetic pathway includes 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.), wherein the product of one encoded enzyme is the substrate for the next enzyme in the synthetic pathway.
As used herein, the term “genetic switch” refers to one or more genetic elements that allow controlled expression of enzymes, e.g., enzymes that catalyze the reactions of isoprenoid biosynthesis pathways. For example, a genetic switch can include one or more promoters operably linked to one or more genes encoding a biosynthetic enzyme, or one or more promoters operably linked to a transcriptional regulator which regulates expression one or more biosynthetic enzymes.
As used herein, the term “heterologous” refers to what is not normally found in nature. The term “heterologous compound” refers to the production of a compound by a cell that does not normally produce the compound, or to the production of a compound at a level not normally produced by the cell. For example, an isoprenoid can be a heterologous compound.
A “heterologous genetic pathway” or a “heterologous biosynthetic pathway” as used herein refer to a genetic pathway that does not normally or naturally exist in an organism or cell.
The term “host cell” as used in the context of this invention refers to a microorganism, such as yeast, and includes an individual cell or cell culture that contains a heterologous vector or heterologous polynucleotide as described herein. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells into which a recombinant vector or a heterologous polynucleotide of the invention has been introduced, including by transformation, transfection, and the like.
As used herein, the terms “isoprenoid”, “isoprenoid compound.” “isoprenoid product,” “terpene,” “terpene compound,” “terpenoid,” and “terpenoid compound” are used interchangeably. They refer to compounds that are capable of being derived from IPP.
As used herein, the term “medium” refers to culture medium and/or fermentation medium.
As used herein, the terms “modified,” “genetically modified,” “recombinant,” and “engineered,” when used to describe a host cell described herein, refer to host cells or organisms that do not exist in nature, host cells or organisms that express compounds, nucleic acids, or proteins at levels that are not expressed by naturally occurring cells or organisms, or host cells or organisms into which a gene or DNA sequence is introduced, regardless of whether the same or similar gene or DNA sequence is already present in the host cell or organism. Thus, a genetically modified host cell can comprise, for example, a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species as the host, but has been incorporated into a host by recombinant methods to form a genetically modified host cell. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA sequence to thereby permit overexpression or modified expression of the gene product of the DNA sequence.
As used herein, the term “naturally occurring” as applied to a nucleic acid, an enzyme, a cell, or an organism, refers to a nucleic acid, enzyme, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and that has not been intentionally modified by a human in the laboratory is naturally occurring. As used herein, the term “non-naturally occurring” means what is not found in nature but is created by human intervention.
As used herein, the phrase “operably linked” refers to a functional linkage between nucleic acid sequences such that the linked promoter and/or regulatory region functionally controls expression of the coding sequence.
As used herein, the terms “overlay.” “oil,” “overlay oil,” or “oil overlay” refer to a biologically compatible hydrophobic, lipophilic, carbon-containing substance including but not limited to geologically-derived crude oil, distillate fractions of geologically-derived crude oil, vegetable oil, algal oil, microbial lipids, or synthetic oils. The oil is neither itself toxic to a biological molecule, a cell, a tissue, or a subject, nor does it degrade (if the oil degrades) at a rate that produces byproducts at toxic concentrations to a biological molecule, a cell, a tissue or a subject.
As used here, “percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as CLUSTAL, BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B. (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid.
The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid as used in the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising. e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages; positive backbones; non-ionic backbones, and non-ribose backbones. Nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. “Polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus, the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. Nucleic acid sequences are presented in the 5′ to 3′ direction unless otherwise specified.
As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
As used herein, the term “production” generally refers to an amount of compound produced by a genetically modified host cell provided herein. In some embodiments, production is expressed as a yield of the compound by the host cell. In other embodiments, production is expressed as a productivity of the host cell in producing the compound.
As used herein, the term “productivity” refers to production of a compound by a host cell, expressed as the amount of non-catabolic compound produced (by weight) per amount of fermentation broth in which the host cell is cultured (by volume) over time (per hour).
As used herein, the term “promoter” refers to a synthetic or naturally derived nucleic acid that is capable of activating, increasing or enhancing expression of a DNA coding sequence, or inactivating, decreasing, or inhibiting expression of a DNA coding sequence. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance or repress expression and/or to alter the spatial expression and/or temporal expression of the coding sequence. A promoter may be positioned 5′ (upstream) of the coding sequence under its control. A promoter may also initiate transcription in the downstream (3′) direction, the upstream (5′) direction, or be designed to initiate transcription in both the downstream (3′) and upstream (5′) directions. The distance between the promoter and a coding sequence to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function. The term also includes a regulated promoter, which generally allows transcription of the nucleic acid sequence while in a permissive environment (e.g., microaerobic fermentation conditions, or the presence of maltose), but ceases transcription of the nucleic acid sequence while in a non-permissive environment (e.g., aerobic fermentation conditions, or in the absence of maltose). Promoters used herein can be constitutive, inducible, or repressible.
As used herein, the term “pyrophosphate” is used interchangeably herein with “diphosphate.”
As used herein, the term “pyrophosphatase” refers to an enzyme having pyrophosphatase activity, i.e., cleaves pyrophosphate from a substrate. For example, TalVeTPP (SEQ ID NO: 19), a phosphatase enzyme from the fungal species Talaromyces verruculosus, has been shown to convert copalyl-pyrophosphate into E-copalol when expressed in the yeast S. cerevisiae or in the bacterium E. coli, and therefore can be a pyrophosphatase.
The term “yield” refers to production of a compound by a host cell, expressed as the amount of compound produced per amount of carbon source consumed by the host cell, by weight.
In one aspect, the disclosure provides for a genetically modified host cell capable of producing gamma-ambryl acetate (GAA), wherein the genetically modified host cell comprises one or more heterologous nucleic acids that each, independently, encodes an enzyme comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12. In some embodiments, the enzyme comprises the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12.
In another aspect, the disclosure provides for a genetically modified host cell capable of producing gamma-ambryl acetate (GAA), wherein the genetically modified host cell comprises one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting manooloxy to GAA. In some embodiments, the enzyme capable of converting manooloxy to GAA is a Baeyer-Villiger monooxygenase (BVMO).
In some embodiments, the genetically modified host cell disclosed herein further comprises one or more heterologous nucleic acids that each, independently, encodes one or more enzymes of a pathway for making GAA. In some embodiments, genetically modified host cell disclosed herein further comprises one or more enzymes comprising the amino acid sequence of SEQ ID NO. 18, 21, 24, 27, 41, 44, 47, or 48. In some embodiments, the genetically modified host cell disclosed herein further comprises one or more of (a) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting one or more IPP, DMAPP, GPP. FPP, or GGPP into GPP, FPP, GGPP, or CPP. (b) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting CPP to E-copalol, (c) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting E-copalol to E-copalal, or (d) one or more heterologous nucleic acids that each, independently, encodes an enzyme capable of converting E-copalal to manooloxy. In some embodiments, genetically modified host cell disclosed herein further comprises a CPP synthase, an Erg20, a GPP synthase, a GGPP synthase, a CPP pyrophosphatase, an alcohol dehydrogenase, or an enal-cleaving enzyme.
In some embodiments, expression of one or more of the enzymes disclosed herein is under the control of a single transcriptional regulator. In some embodiments, expression of one or more of the enzymes disclosed herein is under the control of multiple transcriptional regulators.
In some embodiments, the genetically modified host cell is a yeast cell or a yeast strain. In some embodiments, the yeast cell or the yeast strain is Saccharomyces cerevisiae.
In another aspect, the disclosure provides for a fermentation composition comprising the genetically modified host cell disclosed herein, optionally an overlay, and GAA produced by the genetically modified host cell.
In another aspect, the disclosure provides for a method of producing GAA, comprising culturing the genetically modified host cell disclosed herein in a medium with a carbon source under conditions suitable for making GAA, optionally providing an overlay, and recovering GAA from the genetically modified host cell, the overlay, or the medium.
In another aspect, the disclosure provides for a non-naturally occurring enzyme capable of converting manooloxy to GAA comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12. In an embodiment, the non-naturally occurring enzyme comprises the amino acid sequence of SEQ ID NOS. 3, 6, 9, or 12.
In general, the mevalonate pathway comprises six steps. In the first step, two molecules of acetyl-coenzyme A are enzymatically combined to form acetoacetyl-CoA. An enzyme known to catalyze this step is, for example, acetyl-CoA thiolase (also known as acetyl-CoA acetyltransferase).
In the second step of the MEV pathway, acetoacetyl-CoA is enzymatically condensed with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-COA). An enzyme known to catalyze this step is, for example, HMG-COA synthase.
In the third step, HMG-CoA is enzymatically converted to mevalonate. An enzyme known to catalyze this step is, for example, HMG-COA reductase.
In the fourth step, mevalonate is enzymatically phosphorylated to form mevalonate 5-phosphate. An enzyme known to catalyze this step is, for example, mevalonate kinase.
In the fifth step, a second phosphate group is enzymatically added to mevalonate 5-phosphate to form mevalonate 5-pyrophosphate. An enzyme known to catalyze this step is, for example, phosphomevalonate kinase.
In the sixth step, mevalonate 5-pyrophosphate is enzymatically converted into IPP. An enzyme known to catalyze this step is, for example, mevalonate pyrophosphate decarboxylase.
If IPP is to be converted to DMAPP, then a seventh step is required. An enzyme known to catalyze this step is, for example, IPP isomerase. If the conversion to DMAPP is required, an increased expression of IPP isomerase ensures that the conversion of IPP into DMAPP does not represent a rate-limiting step in the overall pathway.
In general, the DXP pathway comprises seven steps. In the first step, pyruvate is condensed with D-glyceraldehyde 3-phosphate to make 1-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze this step is, for example, 1-deoxy-D-xylulose-5-phosphate synthase.
In the second step, 1-deoxy-D-xylulose-5-phosphate is converted to 2C-methyl-D-erythritol-4-phosphate. An enzyme known to catalyze this step is, for example, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to 4-diphosphocytidyl-2C-methyl-D-erythritol. An enzyme known to catalyze this step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase.
In the fourth step, 4-diphosphocytidyl-2C-methyl-D-erythritol is converted to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. An enzyme known to catalyze this step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.
In the fifth step, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate is converted to 2C-methyl-D-erythritol 2, 4-cyclodiphosphate. An enzyme known to catalyze this step is, for example, 2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase.
In the sixth step, 2C-methyl-D-erythritol 2, 4-cyclodiphosphate is converted to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzyme known to catalyze this step is, for example, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase.
In the seventh step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate is converted into either IPP or its isomer, DMAPP. An enzyme known to catalyze this step is, for example, isopentyl/dimethylallyl diphosphate synthase.
In some embodiments, “cross talk” (or interference) between the host cell's own metabolic processes and those processes involved with the production of IPP as provided herein are minimized or eliminated entirely. For example, cross talk is minimized or eliminated entirely when the host microorganism relies exclusively on the DXP pathway for synthesizing IPP, and a MEV pathway is introduced to provide additional IPP. Such a host organisms would not be equipped to alter the expression of the MEV pathway enzymes or process the intermediates associated with the MEV pathway. Organisms that rely exclusively or predominately on the DXP pathway include, for example, Escherichia coli.
In some embodiments, the host cell produces IPP via the MEV pathway, either exclusively or in combination with the DXP pathway. In other embodiments, a host's DXP pathway is functionally disabled so that the host cell produces IPP exclusively through a heterologously introduced MEV pathway. The DXP pathway can be functionally disabled by disabling gene expression or inactivating the function of one or more of the DXP pathway enzymes.
The pathway from IPP and DMAPP to GAA comprises five steps. The first step involves the production of CPP, which can occur through several possible routes.
One pathway, from IPP and DMAPP to E-copalol, comprises two steps. In the first step, three IPP and one DMAPP are converted to copalyl-pyrophosphate (CPP). Enzymes known to catalyze this step are, for example, chimeric diterpene synthases from Penicillium species. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 16 and 17. In the second step, CPP is converted to E-copalol. An enzyme known to catalyze this step is, for example, a CPP pyrophosphatase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS. 19 and 20.
In another route, two IPP and one DMAPP are converted to FPP. An enzyme known to catalyze this step is, for example, S. cerevisiae Erg20. An illustrative example of a nucleotide sequence includes but is not limited to SEQ ID NO: 40. One IPP and one FPP are then converted to CPP. Enzymes known to catalyze this step are, for example, chimeric diterpene synthases from Penicillium species. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 16 and 17. CPP is then converted to E-copalol. An enzyme known to catalyze this step is, for example, a CPP pyrophosphatase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS. 19 and 20.
Another route involves conversion of two IPP and one DMAPP to form FPP. An enzyme known to catalyze this step is, for example, S. cerevisiae Erg20. An illustrative example of a nucleotide sequence includes but is not limited to SEQ ID NO: 40. One FPP and one IPP are then converted to GGPP. An enzyme known to catalyze this step is, for example, a GGPP synthase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 42 and 43. GGPP is then converted to CPP. An enzyme known to catalyze this step is, for example, a CPP synthase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 45 and 46. Finally, CPP is converted to E-copalol. An enzyme known to catalyze this step is, for example, a CPP pyrophosphatase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS. 19 and 20.
In the second step, CPP is converted to E-copalol. An enzyme known to catalyze this step is, for example, a CPP pyrophosphatase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 19 and 20.
In the third step, E-copalol is converted to E-copalal. An enzyme known to catalyze this step is, for example, an alcohol dehydrogenase. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 22 and 23.
In the fourth step, E-copalal is converted to manooloxy. An enzyme known to catalyze this step is, for example, an enal-cleaving enzyme. Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 25 and 26.
In the fifth step, manooloxy is converted to GAA. Enzymes known to catalyze this step are, for example, some Baeyer-Villiger monooxygenases (BVMO). Illustrative examples of nucleotide sequences include but are not limited to SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10, 11, 13, and 14.
Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding the protein components of the heterologous genetic pathway described herein.
As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons more frequently. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called “codon optimization” or “controlling for species codon bias.”
Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon.
Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. Any one of the polypeptide sequences disclosed herein may be encoded by DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In a similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
In addition, homologs of enzymes useful for the compositions and methods provided herein are encompassed by the disclosure. In some embodiments, two proteins (or a region of the proteins) can be considered homologous when the amino acid sequences have at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art.
The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine(S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. A typical algorithm used for comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer algorithm BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences.
Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in a host cell, for example, a yeast.
In addition, genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed in the host cell. A variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorphs, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., including A. leporis, A. alliaceus, A. brasiliensis, and A. wentii, Neurospora spp., Ustilago spp., Talaromyces spp., including T. amestolkiae, Parastagonospora spp., including P. nodorum, Phacosphacria spp., including P. poagena, Stagonospora spp., Aurcobasidium spp., including A. pullulans, Lepidopterella spp., including L. palustris, or Rhinocladiella spp., including R. mackenziei. Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.
Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes. Generally, analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes. For example, to identify homologous or analogous kinase genes, proteins, or enzymes, techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of an kinase gene/enzyme or by degenerate PCR using degenerate primers designed to amplify a conserved region among kinase genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity, then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence. To identify homologous or similar genes and/or homologous or similar enzymes, analogous genes and/or analogous enzymes or proteins, techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, JGI Phyzome v12.1, BLAST, NCBI RefSeq, UniProt KB, or MetaCYC Protein annotations in the UniProt Knowledgebase may also be used to identify enzymes which have a similar function in addition to the National Center for Biotechnology Information RefSeq database. The candidate gene or enzyme may be identified within the above-mentioned databases in accordance with the teachings herein.
In one aspect, provided herein are host cells comprising at least one enzyme of the isoprenoid biosynthetic pathway. In some embodiments, the isoprenoid biosynthetic pathway contains a genetic regulatory element, such as a nucleic acid sequence, that is regulated by an exogenous agent. In some embodiments, the exogenous agent acts to regulate expression of the heterologous genetic pathway. Thus, in some embodiments, the exogenous agent can be a regulator of gene expression.
In some embodiments, the exogenous agent can be used as a carbon source by the host cell. For example, the same exogenous agent can both regulate production of an isoprenoid compound and provide a carbon source for growth of the host cell. In some embodiments, the exogenous agent is galactose. In some embodiments, the exogenous agent is maltose.
In some embodiments, the genetic regulatory element is a nucleic acid sequence, such as a promoter.
In some embodiments, the genetic regulatory element is a galactose-responsive promoter. In some embodiments, galactose positively regulates expression of the isoprenoid biosynthetic pathway, thereby increasing production of the isoprenoid compound. In some embodiments, the galactose-responsive promoter is a GAL1 promoter. In some embodiments, the galactose-responsive promoter is a GAL10 promoter. In some embodiments, the galactose-responsive promoter is a GAL2, GAL3, or GAL7 promoter. In some embodiments, the host cell lacks the gall gene and is unable to metabolize galactose, but galactose can still induce galactose-regulated genes.
In some embodiments, the galactose regulation system used to control expression of one or more enzymes of the isoprenoid biosynthetic pathway is re-configured such that it is no longer induced by the presence of galactose. Instead, the gene of interest will be expressed unless repressors, which may be maltose in some strains, are present in the medium.
In some embodiments, the genetic regulatory element is a maltose-responsive promoter. In some embodiments, maltose negatively regulates expression of the isoprenoid biosynthetic pathway, thereby decreasing production of the isoprenoid compound. In some embodiments, the maltose-responsive promoter is selected from the group consisting of pMAL1, pMAL2, pMAL11, pMAL12, pMAL31 and pMAL32. The maltose genetic regulatory element can be designed to both activate expression of some genes and repress expression of others, depending on whether maltose is present or absent in the medium.
In some embodiments, the heterologous genetic pathway is regulated by a combination of the maltose and galactose regulons.
In some embodiments, the recombinant host cell does not contain, or expresses a very low level of (for example, an undetectable amount), a precursor required to make the isoprenoid compound. In some embodiments, the precursor is a substrate of an enzyme in the isoprenoid biosynthetic pathway.
In some embodiments, yeast strains useful in the present methods include yeasts that have been deposited with microorganism depositories (e.g. IFO, ATCC, etc.) and belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hyphopichia, Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces, Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozyma, Nadsonia, Nakazawaea, Nematospora, Ogataea, Oosporidium, Pachysolen, Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora, Schizoblastosporion, chizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Sporopachydermia, Stephanoascus, Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces, Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis, and Zygozyma, among others.
In some embodiments, the strain is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (previously called Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorphs (now known as Pichia angusta). In some embodiments, the host microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis.
In a particular embodiment, the strain is Saccharomyces cerevisiae. In some embodiments, the host is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CEN.PK, CEN.PK2, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1. In some embodiments, the strain of Saccharomyces cerevisiae is CEN.PK. In some embodiments, the strain of Saccharomyces cerevisiae is CEN.PK2.
In some embodiments, the strain is a microbe that is suitable for industrial fermentation. In particular embodiments, the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment.
In another aspect, provided are methods of making the modified host cells described herein. In some embodiments, the methods include transforming a host cell with the heterologous nucleic acid constructs described herein which encode the proteins expressed by a heterologous genetic pathway described herein.
In another aspect, methods for producing an isoprenoid compound are described herein. In some embodiments, the method decreases expression of the isoprenoid compound. In some embodiments, the method includes culturing a host cell comprising at least one enzyme of the isoprenoid biosynthetic pathway described herein in a medium comprising an exogenous agent, wherein the exogenous agent decreases the expression of the isoprenoid compound. In some embodiments, the exogenous agent is maltose. In some embodiments, the method results in less than 0.001 mg/L of an isoprenoid compound or a precursor thereof.
In some embodiments, the method is for decreasing expression of an isoprenoid compound or precursor thereof. In some embodiments, the method includes culturing a host cell comprising one or more enzymes of the isoprenoid biosynthetic pathway described herein in a medium comprising an exogenous agent, wherein the exogenous agent decreases the expression of the isoprenoid compound. In some embodiments, the exogenous agent is maltose. In some embodiments, the exogenous agent is maltose. In some embodiments, the method results in the production of less than 0.001 mg/L of an isoprenoid compound or a precursor thereof.
In some embodiments, the method increases the expression of an isoprenoid compound. In some embodiments, the method includes culturing a host cell comprising one or more enzymes of the isoprenoid biosynthetic pathway described herein in a medium comprising the exogenous agent, wherein the exogenous agent increases expression of the isoprenoid compound. In some embodiments, the exogenous agent is galactose. In some embodiments, the method further includes culturing the host cell with the precursor or substrate required to make the isoprenoid compound.
In some embodiments, the method increases the expression of an isoprenoid compound or precursor thereof. In some embodiments, the method includes culturing a host cell comprising a heterologous isoprenoid compound described herein in a medium comprising an exogenous agent, wherein the exogenous agent increases the expression of the isoprenoid compound or a precursor thereof. In some embodiments, the exogenous agent is galactose. In some embodiments, the method further includes culturing the host cell with a precursor or substrate required to make the isoprenoid compound or precursor thereof. In some embodiments, the combination of the exogenous agent and the precursor or substrate required to make the isoprenoid compound or precursor thereof produces a higher yield of the isoprenoid compound than the exogenous agent alone.
Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science. Consideration must be given to appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cell, the fermentation, and the process.
The methods of producing isoprenoid compounds provided herein may be performed in a suitable culture medium in a suitable container, including but not limited to a cell culture plate, a flask, or a fermentor. Further, the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof.
In some embodiments, the culture medium is any culture medium in which a genetically modified microorganism capable of producing a heterologous product can subsist, i.e., maintain growth and viability. In some embodiments, the culture medium is an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients. In some embodiments, the carbon source and each of the essential cell nutrients are added incrementally or continuously to the fermentation medium, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells which convert the carbon source to a biomass.
Suitable conditions and suitable medium for culturing microorganisms are well known in the art. In some embodiments, the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic to select for microorganisms comprising the genetic modifications).
In some embodiments, the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, a complex feedstock, or one or more combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol. Non-limiting examples of a complex feedstock include cane syrup.
The concentration of a carbon source, such as glucose or sucrose, in the culture medium should promote cell growth, but not be so high as to repress growth of the microorganism used. Typically, cultures are run with a carbon source, such as glucose or sucrose, being added at levels to achieve the desired level of growth and biomass. Production of isoprenoid compounds may also occur in these culture conditions, but at undetectable levels (with detection limits being about <0.1 g/l). In other embodiments, the concentration of a carbon source, such as glucose or sucrose, in the culture medium is greater than about 1 g/L, preferably greater than about 2 g/L, and more preferably greater than about 5 g/L. In addition, the concentration of a carbon source, such as glucose or sucrose, in the culture medium is typically less than about 100 g/L, preferably less than about 50 g/L, and sometimes less than about 20 g/L. It should be noted that references to culture component concentrations can refer to both initial and/or ongoing component concentrations. In some cases, it may be desirable to allow the culture medium to become depleted of a carbon source during culture.
Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, vegetable and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen sources, in the culture medium is greater than about 0.1 g/L, preferably greater than about 0.25 g/L, and more preferably greater than about 1.0 g/L. Beyond certain concentrations, however, the addition of a nitrogen source to the culture medium is not advantageous for the growth of the microorganisms. As a result, the concentration of the nitrogen sources, in the culture medium is less than about 20 g/L, preferably less than about 10 g/L and more preferably less than about 5 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culture.
The effective culture medium can contain other compounds such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds can also be present in carbon, nitrogen, or mineral sources in the effective medium or can be added specifically to the medium.
The culture medium can also contain a suitable phosphate source. Such phosphate sources include both inorganic and organic phosphate sources. Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate, and mixtures thereof. Typically, the concentration of phosphate in the culture medium is greater than about 1.0 g/L, preferably greater than about 2.0 g/L, and more preferably greater than about 5.0 g/L. Beyond certain concentrations, however, the addition of phosphate to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of phosphate in the culture medium is typically less than about 20 g/L, preferably less than about 15 g/L, and more preferably less than about 10 g/L.
A suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used. Typically, the concentration of magnesium in the culture medium is greater than about 0.5 g/L, preferably greater than about 1.0 g/L, and more preferably greater than about 2.0 g/L. Beyond certain concentrations, however, the addition of magnesium to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of magnesium in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 3 g/L. Further, in some instances, it may be desirable to allow the culture medium to become depleted of a magnesium source during culture.
In some embodiments, the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate. In such instance, the concentration of a chelating agent in the culture medium is greater than about 0.2 g/L, preferably greater than about 0.5 g/L, and more preferably greater than about 1 g/L. Beyond certain concentrations, however, the addition of a chelating agent to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of a chelating agent in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 2 g/L.
The culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium. Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and mixtures thereof. Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.
The culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride. Typically, the concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg/L to about 2000 mg/L, preferably within the range of from about 20 mg/L to about 1000 mg/L, and more preferably in the range of from about 50 mg/L to about 500 mg/L.
The culture medium can also include sodium chloride. Typically, the concentration of sodium chloride in the culture medium is within the range of from about 0.1 g/L to about 5 g/L, preferably within the range of from about 1 g/L to about 4 g/L, and more preferably in the range of from about 2 g/L to about 4 g/L.
In some embodiments, the culture medium can also include trace metals. Such trace metals can be added to the culture medium as a stock solution of metal salts that, for convenience, can be prepared separately from the rest of the culture medium, with individual components of the stock solution added, for example, at concentrations ranging from 0.3 g/L to 6 g/L. Typically, the amount of such a trace metals solution added to the culture medium is greater than about 1 mL/L, preferably greater than about 5 mL/L, and more preferably greater than about 10 mL/L. Beyond certain concentrations, however, the addition of a trace metals to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the amount of such a trace metals solution added to the culture medium is typically less than about 100 mL/L, preferably less than about 50 mL/L, and more preferably less than about 30 mL/L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution.
The culture medium can include other vitamins, such as biotin, calcium pantothenate, inositol, p-aminobenzoic acid, nicotinic acid, pyridoxine-HCl, and thiamine-HCl. Such vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Beyond certain concentrations, however, the addition of vitamins to the culture medium is not advantageous for the growth of the microorganisms.
The fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous and semi-continuous. In some embodiments, the fermentation is carried out in fed-batch mode. In such a case, some of the components of the medium are depleted during culture, including pantothenate during the production stage of the fermentation. In some embodiments, the culture may be supplemented with relatively high concentrations of such components at the outset, for example, of the production stage, so that growth and/or production is supported for a period of time before additions are required. The preferred ranges of these components can be maintained throughout the culture by making additions as levels are depleted by culture. Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations. Alternatively, once a standard culture procedure is developed, additions can be made at timed intervals corresponding to known levels at particular times throughout the culture. As will be recognized by those in the art, the rate of consumption of nutrient increases during culture as the cell density of the medium increases. Moreover, to avoid introduction of foreign microorganisms into the culture medium, addition is performed using aseptic addition methods, as are known in the art. In addition, a small amount of anti-foaming agent may be added during the culture.
The temperature of the culture medium can be any temperature suitable for growth of the genetically modified cells and/or production of compounds of interest. For example, prior to inoculation of the culture medium with an inoculum, the culture medium can be brought to and maintained at a temperature in the range of from about 20° C. to about 45° C., preferably to a temperature in the range of from about 25° C. to about 40° C. and more preferably in the range of from about 28° C. to about 32° C.
The pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium. Preferably, the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.
In some embodiments, the carbon source concentration, such as the glucose concentration, of the culture medium is monitored during culture. Glucose or sucrose concentration of the culture medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the culture medium. As stated previously, the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g/L and can be determined readily by trial. Accordingly, when glucose is used as a carbon source the glucose is preferably fed to the fermenter and maintained in the range of from about 1 g/L to about 100 g/L, or in the range of from about 2 g/L to about 50 g/L, or in the range of from about 5 g/L to about 20 g/L. Alternatively, the glucose concentration in the culture medium is maintained below detection limits. Although the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by addition of aliquots of the original culture medium. The use of aliquots of the original culture medium may be desirable because the concentrations of other nutrients in the medium (e.g. the nitrogen and phosphate sources) can be maintained simultaneously. Likewise, the trace metals concentrations can be maintained in the culture medium by addition of aliquots of the trace metals solution.
The following examples are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
Each DNA construct was integrated into Saccharomyces cerevisiae (CEN.PK2) with standard molecular biology techniques in an optimized lithium acetate (LiAc) transformation. Briefly, cells were grown overnight in standard liquid culture medium at 30° C. with shaking (200 rpm), diluted to an OD600 of 0.1 in fresh medium, and grown to an OD600 of 0.6-0.8. For each transformation, 5 mL of culture were harvested by centrifugation, washed in 5 mL of sterile water, spun down again, resuspended in 1 mL of 100 mM LiAc, and transferred to a microcentrifuge tube. Cells were spun down (13,000×g) for 30 seconds, the supernatant was removed, and the cells were resuspended in a transformation mix of 240 μL 50% PEG, 36 μL 1 M LiAc, 10 μL boiled salmon sperm DNA, and 74 μL of donor DNA (˜1 μg). Following a heat shock at 42° C. for 40 minutes, cells were centrifuged and suspended in liquid culture medium for overnight recovery at 30° C. with shaking (200 rpm) before plating on solid agar selective medium. DNA integration was confirmed by yeast colony PCR with primers specific to the integrations.
Certain fungal Baeyer-Villiger monooxygenase (BVMO) enzymes have been shown to oxygenate the cyclic isoprenoid molecule manooloxy to form gamma-ambryl acetate (GAA). For example, AspWeBVMO (SEQ ID NO: 15), a BVMO enzyme from the fungal species Aspergillus wentii, converts manooloxy into GAA when expressed in the yeast S. cerevisiae.
A manooloxy production strain was created from an S. cerevisiae base strain (CEN.PK2) by integrating and expressing codon-optimized versions of genes encoding the following heterologous proteins, all under control of strong S. cerevisiae promoters: a synthase PvCPS to convert IPP and DMAPP to CPP (SEQ ID NO: 17), a CPP pyrophosphatase TalVeTPP to convert CPP to E-copalol (SEQ ID NO: 20), an alcohol dehydrogenase to convert E-copalol to E-copalal (SEQ ID NO: 23), and an enal-cleaving enzyme to convert E-copalal to manooloxy (SEQ ID NO: 26). This test strain was then used to identify and to rank novel BVMO enzymes that have the ability to convert manooloxy into GAA.
GAA production strains were generated by integrating candidate BVMO enzymes identified from public sequence databases based on similarity to known manooloxy oxygenases such as AspWeBVMO. DNA sequences were codon-optimized for expression in S. cerevisiae, and integrated into the test strain described above under control of a strong S. cerevisiae promoter. To serve as a benchmark, another strain was constructed with a codon-optimized gene expressing AspWeBVMO (SEQ ID NO:14). The ability of novel BVMO candidates to convert manooloxy into GAA was determined by quantifying GAA production from cultured yeast strains.
Yeast were inoculated into 96-well microtiter plates containing 120 μL per well Bird Seed Media (100 ml/L Bird Batch (potassium phosphate 80 g/L, ammonium Sulfate 150 g/L, magnesium sulfate 61.5 g/L), 5 ml/L Trace Metal Solution (0.5M EDTA 160 mL/L, zinc sulfate heptahydrate 11.5 g/L, copper sulfate 0.64 g/L, manganese (II) chloride 0.64 g/L, cobalt (II) chloride hexahydrate 0.94 g/L, sodium molybdate 0.96 g/L, iron (II) sulfate 5.6 g/L, calcium chloride dihydrate 5.8 g/L), 12 ml/L Birds Vitamins 2.0 (biotin 0.05 g/L, p-aminobenzoic acid 0.2 g/L, calcium pantothenate 1 g/L, nicotinic acid 1 g/L, myoinositol 25 g/L, thiamine HCl 1 g/L, pyridoxine HCl 1 g/L), and succinic acid 6 g/L; pH 5), with 4% sucrose, and a hydrophobic isopropyl myristate overlay added at 25% of aqueous volume. Microtiter plates were sealed with gas-permeable membranes and cultured at 30° C. in a high-capacity microtiter plate incubator shaking at 1000 rpm and 80% humidity for 3 days, by which time cultures had reached carbon exhaustion.
To quantify the conversion of manooloxy to GAA in cultures of strains that contain different candidate BVMO enzymes, manooloxy and GAA were measured by gas chromatography (GC). The performance of different candidate enzymes was ranked relative to the AspWeBVMO benchmark strain based on the absolute amount of GAA produced and on the amount of GAA product relative to manooloxy substrate. To assess titers, cultures from 96-well plates were extracted with 10 volumes of ethyl acetate (relative to aqueous volume) by shaking at 1000 rpm for 30 seconds at room temperature, and extractant was separated by centrifugation (2000 rpm for 5 minutes) and analyzed on an Agilent 7890A with flame ionization detection (GC-FID) along with analytical standards. The following ramped temperature program with constant flow at 1.4 mL/min was used for analysis:
Native enzymes from four different fungal species demonstrated improved activity in conversion of manooloxy to GAA relative to AspWeBVMO when expressed in the S. cerevisiae manooloxy-producing test strain.
Aspergillus wentii
Lepidopterella palustris CBS
Aspergillus alliaceus
Rhinocladiella mackenziei
Aspergillus brasiliensis CBS
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.
All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/073758 | 7/15/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63222590 | Jul 2021 | US |
