Patent Application
20190071474
The sequence listing submitted herewith, entitled “15-1649-WO_SequenceListing_ST25.txt” and 713 kb in size, is incorporated by reference in its entirety.
This disclosure relates to recombinant production of gibberellin compounds and gibberellin precursors in recombinant hosts. In particular, this disclosure relates to production of gibberellin A3 (i.e., GA3) in recombinant hosts.
Gibberellins are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development during a plant's life cycle, including, but not limited to, seed germination, stem elongation, sex expression, flowering, formation of fruits, and senescence. Gibberellin structure is shown in
In plants, fungi, and bacteria, gibberellins are synthesized from kaurenoic acid in a stepwise fashion, wherein a series of functional group additions and oxidations are performed by cytochrome P450 monooxygenases (P450s) and 2-oxoglutarate-dependent dioxygenases (2-ODDs). See,
In plants, the P450 enzyme involved is kaurenoic acid oxidase (KAO) and the 2-ODD enzymes are GA oxidases (e.g., GA20ox, GA7ox, etc.). In fungi, the P450 enzymes P450-1, P450-2, and P450-3 are responsible for the majority of the gibberellin synthesis pathway, while GA4 desaturase (DES) is the only 2-ODD enzyme involved. See, Yamaguchi, Annu. Rev. Plant Biol. 59:225-51 (2008); Bömke and Tudzynski, Phytochemistry 70:1876-93 (2009). In bacteria, P450 enzymes perform the majority of gibberellin biosynthesis. See, Bottini et al., 2004, Appl. Microbiol. Biotechnol. 65:497-503.
GA3 (gibberellic acid), is used commercially for a variety of purposes, including inducing seed germination, inducing flowering, and increasing fruit size. Because plants produce only minute amounts of GA3, the hormone is produced industrially by submerged fermentation using the fungus Gibberella fujikuroi (also known as Fusarium fujikuroi.) F. fujikuroi is not a preferred production host due to slow growth compared to other production hosts; an F. fujikuroi fermentation typically can last up 9 days, while a Saccharomyces cerevisiae fermentation usually is completed in 4-5 days. See Uthandi et al., 2009, Journal of Scientific & Industrial Research 69:211-4 and Rodrigues et al., 2009, Braz. Arch. Biol. Tech. 52(Special No.):181-8. As production, recovery, and purification of GA3 and other gibberellins have proven to be costly, there remains a need for a recombinant production system that can accumulate high yields of desired gibberellins, such as GA3, GA4, GA7, or GA1, in a more cost-effective manner.
It is against the above background that the present invention provides certain advantages and advancements over the prior art.
Although this invention as disclosed herein is not limited to specific advantages or functionalities, the invention provides a recombinant host cell, comprising:
wherein the recombinant host cell is capable of producing a gibberellin precursor and/or a gibberellin compound.
In one aspect of the recombinant host cell disclosed herein, the gene encoding the first P450 polypeptide encodes a kaurenoic acid oxidase (KAO) polypeptide or a cytochrome P450 monooxygenase-1 (P450-1) polypeptide.
In one aspect of the recombinant host cell disclosed herein, the gene encoding the first P450 polypeptide comprises:
In one aspect of the recombinant host cell disclosed herein,
In one aspect of the recombinant host cell disclosed herein, the gene encoding the second P450 polypeptide comprises:
In one aspect of the recombinant host cell disclosed herein, the gene encoding the 2-ODD polypeptide comprises:
In one aspect of the recombinant host cell disclosed herein,
The invention further provides a recombinant host cell comprising:
wherein the recombinant host cell is capable of producing a gibberellin precursor and/or a gibberellin compound.
The invention further provides a recombinant host cell, comprising:
wherein the recombinant host cell is capable of producing a gibberellin precursor and/or a gibberellin compound.
The invention further provides a recombinant host cell comprising a gene encoding a kaurenoic acid oxidase (KAO) polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:62, SEQ ID NO:60, or SEQ ID NO:152, at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:58 or SEQ ID NO:68, at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:64, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:74;
wherein the recombinant host cell is capable of producing gibberellin precursor and/or a gibberellin compound.
In one aspect of the recombinant host cell disclosed herein, the recombinant host cell further comprises:
The invention further provides a recombinant cell host, comprising:
wherein the recombinant host cell is capable of producing a gibberellin precursor and/or a gibberellin compound.
The invention further provides a recombinant host cell, comprising:
wherein the recombinant host cell is capable of producing a gibberellin precursor and/or a gibberellin compound.
In one aspect of the recombinant host cell disclosed herein, the recombinant host cell further comprises:
In one aspect of the recombinant host cell disclosed herein,
In one aspect of the recombinant host cell disclosed herein, the recombinant host cell further comprises:
In one aspect of the recombinant host cell disclosed herein,
In one aspect of the recombinant host cell disclosed herein, expression of the recited genes increases the portion of the gibberellin precursor and/or the gibberellin compound produced by the recombinant host cell by at least about 10%, 25%, 50%, 75%, 80%, 90%, 95%, 100% or more.
In one aspect of the recombinant host cells disclosed herein, the gibberellin compound comprises GA1, GA3, GA4, GA5, GA7, GA9, GA12, GA13, GA14, GA15, GA19, GA20, GA24, GA25, GA36, GA37, GA44, GA53, and/or GA110.
In one aspect of the recombinant host cells disclosed herein, the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell or a bacterial cell.
The invention further provides a method of producing a gibberellin precursor and/or a gibberellin compound in a cell culture, comprising growing the recombinant host cell disclosed herein in a cell culture, under conditions in which the genes are expressed;
wherein the gibberellin precursor and/or the gibberellin compound is produced by the recombinant host cell.
In one aspect, the method disclosed herein further comprises isolating the gibberellin precursor and/or the gibberellin compound from the cell culture.
In one aspect of the method of producing a gibberellin precursor and/or gibberellin compound in a cell culture, the isolating step comprises:
In one aspect, the method disclosed herein further comprises recovering the gibberellin precursor and/or the gibberellin compound.
In one aspect, the method disclosed herein further comprises
In one aspect of the methods disclosed herein:
In one aspect, the method disclosed herein further comprises a step of converting GA4 to GA1 catalyzed by a third P450 polypeptide.
In one aspect of the method disclosed herein, the third P450 polypeptide comprises a P450-3 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:186; or a GA13ox-1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:98.
In one aspect, the method disclosed herein further comprises:
In one aspect of the method disclosed herein:
In one aspect, the method disclosed herein further comprises:
In one aspect of the method disclosed herein:
In one aspect, the method disclosed herein further comprises a step of converting GA4 to GA1 catalyzed by a second P450 polypeptide.
In one aspect of the method disclosed herein, the second P450 polypeptide comprises a P450-3 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:186; or a GA13ox-1 polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:98.
In one aspect, the method disclosed herein further comprises:
In one aspect of the method disclosed herein:
In one aspect of the method disclosed herein the recombinant host cell is grown in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the gibberellin precursor and/or the gibberellin compound.
In one aspect of the methods disclosed herein, the gibberellin compound comprises GA3 and its precursors, metabolites, or related compounds, including: GA1, GA4, GA5, GA7, GA9, GA12, GA13, GA14, GA15, GA19, GA20, GA24, GA25, GA36, GA37, GA44, GA53, and/or GA110.
In one aspect of the methods disclosed herein, the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell or a bacterial cell.
The invention further provides a cell culture, comprising the recombinant host cell disclosed herein, the cell culture further comprising:
wherein one or more gibberellin precursors and/or the gibberellin compounds are present at a concentration of at least 100 mg/liter of the cell culture.
The invention further provides a cell lysate from the recombinant host cell disclosed herein and grown in the cell culture, comprising:
wherein one or more gibberellin precursors and/or the gibberellin compounds are present at a concentration of at least 100 mg/liter of the cell culture.
These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids.
It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, Calif.).
As used herein, the terms “polynucleotide,” “nucleotide,” “oligonucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
As used herein, the terms “microorganism,” “microorganism host,” “microorganism host cell,” “recombinant host,” and “recombinant host cell” can be used interchangeably. As used herein, the term “recombinant host” is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. The term “transformant(s)” is intended to refer a host to which at least one DNA sequence has been introduced. Such DNA sequences for “recombinant host” and “transformant(s)” include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, for example bacteria, fungi or yeast.
As used herein, the term “recombinant gene” refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. 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 to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in Saccharomyces cerevisiae (S. cerevisiae).
As used herein, the term “engineered biosynthetic pathway” refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
As used herein, the term “endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some embodiments, the endogenous gene is a yeast gene. In some embodiments, the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C. In some embodiments, an endogenous yeast gene is overexpressed. As used herein, the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. In some embodiments, an endogenous yeast gene, for example ADH, is deleted. As used herein, the terms “deletion,” “deleted,” “knockout,” and “knocked out” can be used interchangeably to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae.
As used herein, the terms “heterologous sequence” and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
A “selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Non-limiting examples of a selectable marker can include a URA3 marker and a NatMx maker. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art. Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, PCR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see e.g., U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
As used herein, the terms “variant” and “mutant” are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
As used herein, the term “inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
As used herein, the term “gibberellin” refers to a diterpene plant hormone having the structure of the molecule shown in Formula I and
As used herein, the term “gibberellin precursor” refers to intermediate compounds in a gibberellin biosynthetic pathway. Gibberellin precursors include, but are not limited to, GGPP, ent-copalyl-diphosphate, ent-kaurene, ent-kaurenoic acid, and ent-kaurenoic acid-7-α-OH kaurenoic acid. See, e.g.,
In some aspects, gibberellins and gibberellin precursors are accumulated in an ent-kaurenoic acid-producing host. Recombinant ent-kaurenoic acid-producing and terpene-producing Saccharomyces cerevisiae (S. cerevisiae) strains are described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which has been incorporated by reference herein in its entirety. Methods of producing terpenes in recombinant hosts, by whole cell bio-conversion, and in vitro are also described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
In some embodiments, gibberellins and/or gibberellin precursors are produced in vivo through expression of one or more enzymes involved in a gibberellin biosynthetic pathway in a recombinant host. For example, an ent-kaurenoic acid-producing recombinant host expressing one or more of a gene encoding a cytochrome P450 (P450) monooxygenase polypeptide, a gene encoding a cytochrome P450 reductase (CPR) polypeptide, and a gene a 2-ODD polypeptide can accumulate a gibberellin or gibberellin precursor in vivo. See, e.g.,
In some embodiments, gibberellins and/or gibberellin precursors are produced through contact of a gibberellin precursor with one or more enzymes involved in the gibberellin pathway in vitro. For example, contacting GA7 with a cytochrome P450 polypeptide can result in production of GA3 in vitro. In some embodiments, a gibberellin is produced through contact of a gibberellin precursor with one or more enzymes involved in the gibberellin pathway in vitro. For example, contacting ent-kaurene with a KO enzyme can result in production of ent-kaurenoic acid in vitro.
In some embodiments, a gibberellin or gibberellin precursor is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host cell expressing one or more enzymes involved in the gibberellin pathway takes up and modifies a gibberellin precursor in the cell; following modification (e.g., addition of a double bond or oxidation) in vivo, a gibberellin remains in the cell and/or diffuses or is excreted into the culture medium. For example, a host cell expressing a gene encoding a cytochrome P450 monooxygenase polypeptide can take up GA7 and oxidize C13 of GA7 in the cell; following such a modification in vivo, GA3 can be excreted into the culture medium. In some embodiments, the cell can be permeabilized to take up a substrate to be modified or to excrete a modified product.
In some embodiments, one or more gibberellin precursors and/or one or more gibberellins are produced by co-culturing of two or more hosts. In some embodiments, one or more hosts, each expressing one or more enzymes involved in the gibberellin pathway, produce one or more gibberellin precursors and/or one or more gibberellins. For example, a host comprising a GGPPS, an CDPS, and/or a KO and a host comprising a cytochrome P450 monooxygenase, a cytochrome P450 reductase, and/or a 2-ODD produce one or more gibberellins.
In some aspects, a host comprises a heterologous gene encoding a GGPPS polypeptide. In some embodiments, the GGPPS polypeptide is a GGPPS polypeptide having the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:134, or SEQ ID NO:178. The GGPPS polypeptide can catalyze conversion of farnesyl diphosphate (FPP) to GGPP.
In some aspects, a host comprises a heterologous gene encoding a CDPS polypeptide. In some embodiments, the CDPS polypeptide is a CDPS polypeptide having the amino acid sequence set forth in SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, or SEQ ID NO:180 or a bi-functional a CDPS polypeptide having the amino acid sequence set forth in SEQ ID NO:104, SEQ ID NO:227 or SEQ ID NO:229. The CDPS polypeptide can catalyze conversion of GGPP to ent-copalyl pyrophosphate. In some embodiments, the bi-functional CDPS polypeptide of SEQ ID NO:104 further comprises a P571S and/or L654P substitution. In some embodiments, a host comprising the mutant CDPS polypeptide accumulates greater levels of gibberellins, as compared to a host that does not comprise a gene encoding a mutant CDPS polypeptide.
In some aspects, a host comprises a heterologous gene encoding a KS polypeptide. In some embodiments, the KS polypeptide is a KS polypeptide having the amino acid sequence set forth in SEQ ID NO:102 or SEQ ID NO:106. The KS polypeptide can catalyze conversion of ent-copalyl pyrophosphate to ent-kaurene.
In some aspects, a host comprises a heterologous gene encoding a KO polypeptide. In some embodiments, the KO polypeptide is a KO polypeptide having the amino acid sequence set forth in SEQ ID NO:82, SEQ ID NO:164, SEQ ID NO:170, or SEQ ID NO:172. The KO polypeptide can catalyze conversion of ent-kaurene to ent-kaurenoic acid.
In some aspects, a host comprises a gene encoding a KAO polypeptide. The KAO polypeptide can be a plant-derived KAO polypeptide. In some embodiments, the KAO polypeptide is a KAO polypeptide having the amino acid sequence set forth in SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:88, SEQ ID NO:90, or SEQ ID NO:146. The KAO polypeptide can catalyze, for example, conversion of ent-kaurenoic acid to ent-7α-OH kaurenoic acid, ent-7α-OH kaurenoic acid to GA12 aldehyde, GA12 aldehyde to GA12, and GA12 aldehyde to GA14 aldehyde. See, e.g.,
In some embodiments, a cytochrome B5 polypeptide (i.e., a cytochrome B5 polypeptide of SEQ ID NO:160) and/or a cytochrome B5 reductase polypeptide (i.e., a cytochrome B5 reductase polypeptide of SEQ ID NO:2) increases activity of a KAO polypeptide and/or a cytochrome P450 polypeptide. In some aspects, increased activity of a KAO polypeptide is evidenced by increased levels of GA14 and GA3 in an S. cerevisiae strain comprising a gene encoding a cytochrome B5 polypeptide and a gene encoding a cytochrome b5 reductase polypeptide. See Example 2 and
In some aspects, a host comprises a gene encoding a P450-1 polypeptide. The P450-1 polypeptide can be a fungus-derived P450-1 polypeptide. In some embodiments, the P450-1 polypeptide is a P450-1 polypeptide having the amino acid sequence set forth in SEQ ID NO:74, SEQ ID NO:88, SEQ ID NO:90, or SEQ ID NO:146. The P450-1 polypeptide can catalyze conversion of ent-kaurenoic acid to ent-7α-OH kaurenoic acid, ent-7α-OH kaurenoic acid to GA12 aldehyde, and GA12 aldehyde to GA14 aldehyde. In some aspects, a P450-1 polypeptide can have KAO and GA3ox activity. See Example 8. The fungal KAO enzymes (e.g., S. manihoticola KAO4 polypeptide (SEQ ID NO:73, SEQ ID NO:74) and G. fujikuroi KAO1 polypeptide (SEQ ID NO:89, SEQ ID NO:90) also have GA3ox activity.
In some aspects, a host comprises a gene encoding a GA 20-oxidase (GA20ox) polypeptide. The GA20ox polypeptide can be a plant-derived GA20ox polypeptide. In some embodiments, the GA20ox polypeptide comprises a GA20ox polypeptide having the amino acid sequence set forth in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:40, or SEQ ID NO:42. The GA20ox polypeptide is a 2-ODD polypeptide and can catalyze conversion of GA14 to GA4, GA12 to GA15, GA24 to GA9, GA53 to GA44, and GA44 to GA19. See
In other embodiments, a host comprises a GA 7-oxidase (GA7ox) and/or a GA 3-oxidase (GA3ox). GA7ox and GA3ox polypeptides can be plant-derived 2-ODD polypeptides. In some embodiments, the GA7ox polypeptide comprises a GA7ox polypeptide having the amino acid sequence set forth in SEQ ID NO:16 or SEQ ID NO:162. In some embodiments, the GA3ox polypeptide comprises a GA3ox polypeptide having the amino acid sequence set forth in SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:36, or SEQ ID NO:44.
In some embodiments, a host comprises a GA 13-oxidase (GA13ox). A GA13ox polypeptide can be a plant-derived GA13ox polypeptide. In some embodiments, the GA13ox polypeptide comprises a GA13ox polypeptide having the amino acid sequence set forth in SEQ ID NO:72, SEQ ID NO:78, or SEQ ID NO:98. In some embodiments, a cytochrome B5 polypeptide (i.e., a cytochrome B5 polypeptide of SEQ ID NO:160) and/or a cytochrome B5 reductase polypeptide (i.e., a cytochrome B5 reductase polypeptide of SEQ ID NO:2) increases activity of a GA13ox polypeptide. In some embodiments, the GA13ox polypeptide can catalyze conversion of GA9 to GA20. See
In some aspects, a host comprises a gene encoding a P450-2 polypeptide. The P450-2 polypeptide can be a fungus-derived P450-2 polypeptide. In some embodiments, the P450-2 polypeptide comprises a P450-2 polypeptide having the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:70, SEQ ID NO:80, SEQ ID NO:94, SEQ ID NO:142, SEQ ID NO:233, SEQ ID NO:235, or SEQ ID NO:237. The P450-2 polypeptide can catalyze conversion of GA14 to GA4 and conversion of GA12 to GA9. See
In some aspects, a host comprises a gene encoding a P450-3 polypeptide. The P450-3 polypeptide can be a fungus-derived P450-3 polypeptide. In some embodiments, the P450-3 polypeptide comprises a P450-3 polypeptide having the amino acid sequence set forth in SEQ ID NO:46, SEQ ID NO:144, SEQ ID NO:184, or SEQ ID NO:186. The P450-3 polypeptide can catalyze conversion of GA4 to GA1 or GA7 to GA3. See
In some embodiments, a host comprises a gene encoding a GA4 desaturase (DES) polypeptide. The DES polypeptide can be a fungus-derived DES polypeptide. In some embodiments, the DES polypeptide comprises a DES polypeptide having the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. In some aspects, the DES polypeptide of SEQ ID NO:22 and/or the DES polypeptide of SEQ ID NO:26 comprises an L233P substitution. The DES polypeptide is a 2-ODD polypeptide and can catalyze conversion of GA4 to GA7. See
In some embodiments, a host comprises a gene encoding a cytochrome B5 polypeptide and/or a gene encoding a cytochrome B5 reductase polypeptide. In some aspects, a cytochrome B5 reductase provides electrons to a P450 monooxygenase through cytochrome B5. In some aspects, the cytochrome B5 electron transport system assists a cytochrome P450 reductase by supplying an electron of the catalytic cycle or by acting as an allosteric activator. See, e.g., Troncoso et al., 2008, Phytochemistry 69(3):672-83. In some embodiments, the cytochrome B5 polypeptide comprises a cytochrome B5 polypeptide having the amino acid sequence set forth in SEQ ID NO:160. In some embodiments, the cytochrome B5 reductase polypeptide comprises a cytochrome B5 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. See Example 2.
In some embodiments, a host comprises a CYP112 polypeptide. In some embodiments, the CYP112 polypeptide comprises a CYP112 polypeptide having the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:124, or SEQ ID NO:128. The CYP112 polypeptide can catalyze conversion of GA12 to GA15, GA15 to GA24, GA24 to GA9, and GA14 to GA4. See
In some embodiments, a host comprises one or more heterologous genes encoding one or more alcohol dehydrogenase (ADH) polypeptides. The ADH polypeptide can be an ADH polypeptide having the amino acid sequence set forth in SEQ ID NO:112, SEQ ID NO:116, or SEQ ID NO:118. See
In some embodiments, a host comprising CDPS-KS bifunctional polypeptides can be comparatively tested in a host inserted with CytB5-1 and CytB5red-1. The host may then be transformed with CPR12 (SEQ ID NO:167 which encodes SEQ ID NO:168), RsKO_GA (SEQ ID NO:169 which encodes SEQ ID NO:170), GGPPS7 (SEQ ID NO:176 and SEQ ID NO:178), KO1 (SEQ ID NO:171 which encodes SEQ ID NO:172), and either CDPS-KS6+KS5 (SEQ ID NO:101 which encodes SEQ ID NO:102; and SEQ ID NO:181 which encodes SEQ ID NO:182), CDPS-KS6 (SEQ ID NO:101 which encodes SEQ ID NO:102), CDPS-KS4 (SEQ ID NO:226 which encodes SEQ ID NO:227), or CDPS-KS9 (SEQ ID NO 228 which encodes SEQ ID NO:229). See Example 3 and Table 6. In some aspects, the CDPS-KS activity converts GGPPS to kaurenoic acid.
In some embodiments, a host may comprise KO1 (SEQ ID NO:171 (nt) and SEQ ID NO:172 (aa) and CPR19 (SEQ ID NO:193 (nt) and SEQ ID NO:194 (aa)) and may be transformed with CDPS-KS6 (SEQ ID NO:101), KS5 (SEQ ID NO:181), GGPPS7 (SEQ ID NO:177), KO1 (SEQ ID NO:171), KAO and CPR genes using USER™ based DNA assembler vectors and NatMx marker. The host may co-express KAO-3/CPR19 polypeptides (SEQ ID NO:230 and SEQ ID NO:193), KAO-4/CPR17 (SEQ ID NO:73 and SEQ ID NO:187) or CPR19 (SEQ ID NO:193) polypeptides, or KAO-5/CPR12 (SEQ ID NO:61 and SEQ ID NO:167) or CPR19 polypeptides (for example, SEQ ID NO:193). See Example 4,
In some embodiments, a host may comprise FfCytB5-1 (SEQ ID NO:159 (nt) and SEQ ID NO:160 (aa)), FfCytB5red-1 (SEQ ID NO:01 (nt) and SEQ ID NO:02 (aa)), CPR19 (SEQ ID NO:193 (nt) and SEQ ID NO:194 (aa)), RsKO-GA (SEQ ID NO:169 (nt) and SEQ ID NO:170 (aa)), KS5 (SEQ ID NO:181 (nt) and SEQ ID NO:182 (aa)), tCDPS5 (SEQ ID NO:179 (nt) and SEQ ID NO:180 (aa)), GGPPS-7 (SEQ ID NO:177 (nt) and SEQ ID NO:178 (aa)), and KO1 (SEQ ID NO:171 (nt) and SEQ ID NO:172 (aa)) and be transformed with P450-3-1 (SEQ ID NO:45), P450-2-4 (SEQ ID NO:141), P450-3-4 (SEQ ID NO:185), DES-1 (SEQ ID NO:25), and either KAO1 (SEQ ID NO:89), KAO3 (SEQ ID NO:145), KAO4 (SEQ ID NO:73) or KAO5 (SEQ ID NO:61). See Example 4,
In some embodiments, a host may be inserted with P450-3-4 (SEQ ID NO:141(nt) and SEQ ID NO:142 (aa)), KO1 (SEQ ID NO:170 (nt) and SEQ ID NO:171 (aa)), GGPPS-7 (SEQ ID NO:177 (nt) and SEQ ID NO:178 (aa)), CDPS-KS6 (SEQ ID NO:101 (nt) and SEQ ID NO:102 (aa)), KAO4 (SEQ ID NO:73 (nt) and SEQ ID NO:74 (aa)), FfCytB5-1 (SEQ ID NO:159 (nt) and SEQ ID NO:160 (aa)), CPR1 (SEQ ID NO:165 (nt) and SEQ ID NO:166 (aa)), CPR19 (SEQ ID NO:193 (nt) and SEQ ID NO:194 (aa)), and various P450-2 genes: P450-2-1 (SEQ ID NO:79 (nt) and SEQ ID NO:80 (aa)), P450-2-8 (SEQ ID NO:232 (nt) and SEQ ID NO:233 (aa)), P450-2-9 (SEQ ID NO:234 (nt) and SEQ ID NO:235 (aa)), and P450-2-10 (SEQ ID NO:236 (nt) and SEQ ID NO:237 (aa)). See Example 5, Table 9, and
In some embodiments, P450-2 genes may be introduced by integration into a host using a USER™ cloning based vector system using the URA3 selection marker. P450-2 genes integrated may be selected from SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:80, and SEQ ID NO:141. See Example 5, Table 10, and
In some embodiments, an S. cerevisiae strain (strain “N”) comprising a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179, SEQ ID NO:180), a gene encoding a KS polypeptide (SEQ ID NO:181, SEQ ID NO:182), a first gene encoding a KO polypeptide (SEQ ID NO:171, SEQ ID NO:172), a second gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a CPR polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:195, SEQ ID NO:196), a gene encoding a Gibberellin fujikuroi P450-2-1 polypeptide (SEQ ID NO:79, SEQ ID NO:80), a gene encoding a Gibberellin fujikuroi P450-3-4 polypeptide (SEQ ID NO:185, SEQ ID NO:186), a gene encoding an S. manihoticola KAO4 polypeptide (SEQ ID NO:73, SEQ ID NO:74), a gene encoding a G. fujikuroi DES-1 polypeptide (SEQ ID NO:25, SEQ ID NO:26), a gene encoding a G. fujikuroi cytochrome B5 polypeptide (SEQ ID NO:159, SEQ ID NO:160), and a gene encoding a G. fujikuroi cytochrome B5 reductase polypeptide (SEQ ID NO:1, SEQ ID NO:2) accumulate gibberellins, including, but not limited to, GA3, GA4, GA12, GA14, and GA17. See Example 2; Tables 2 and 4; and
In some embodiments, an S. cerevisiae strain (strain “A”) comprising a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179, SEQ ID NO:180), a gene encoding a KS polypeptide (SEQ ID NO:181, SEQ ID NO:182), a first gene encoding a KO polypeptide (SEQ ID NO:171, SEQ ID NO:172), a second gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a CPR polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:195, SEQ ID NO:196), a gene encoding a Gibberellin fujikuroi P450-2-1 polypeptide (SEQ ID NO:79, SEQ ID NO:80), a gene encoding a Gibberellin fujikuroi P450-3-4 polypeptide (SEQ ID NO:185, SEQ ID NO:186), a gene encoding an S. manihoticola KAO4 polypeptide (SEQ ID NO:73, SEQ ID NO:74), a gene encoding a G. fujikuroi DES-1 polypeptide (SEQ ID NO:25, SEQ ID NO:26), and a gene encoding an A. niger CPR12 polypeptide (SEQ ID NO:157, SEQ ID NO:158) accumulates gibberellins, including, but not limited to, GA3, GA4, GA12, GA13, GA14, GA25. See Example 2; Tables 3 and 4; and
In some embodiments, expression of ORF1 (SEQ ID NO:153, SEQ ID NO:154), ORF2 (SEQ ID NO:155, SEQ ID NO:156), AIdDH (SEQ ID NO:201, SEQ ID NO:202), ADH (SEQ ID NO:109, SEQ ID NO:110), ANK (SEQ ID NO:210, SEQ ID NO:225) and/or smt (SEQ ID NO:222, SEQ ID NO:209), which are clustered with various gibberellin pathway genes in G. fujikuroi, can improve turnover of gibberellin-producing S. cerevisiae strains described herein. See e.g., Bömke et al., 2009, Phytochemistry, 70(15-16):1876-93.
In some embodiments, an S. cerevisiae strain (strain “F”) comprising a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179, SEQ ID NO:180), a gene encoding a KS polypeptide (SEQ ID NO:181, SEQ ID NO:182), a first gene encoding a KO polypeptide (SEQ ID NO:171, SEQ ID NO:172), a second gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a CPR polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:195, SEQ ID NO:196), a gene encoding an A. thaliana GA20ox-4 polypeptide (SEQ ID NO:39, SEQ ID NO:40), a gene encoding a G. fujikuroi P450-3-4 polypeptide (SEQ ID NO:185, SEQ ID NO:186), a gene encoding an S. manihoticola KAO4 polypeptide (SEQ ID NO:73, SEQ ID NO:74), a gene encoding a G. fujikuroi DES-1 polypeptide (SEQ ID NO:25, SEQ ID NO:26), and a gene encoding an A. niger CPR16 polypeptide (SEQ ID NO:157, SEQ ID NO:158) accumulates gibberellins, including, but not limited to, GA3, GA4, GA12, and GA14. See Example 2,
In some embodiments, an S. cerevisiae strain comprising a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179, SEQ ID NO:180), a gene encoding a KS polypeptide (SEQ ID NO:181, SEQ ID NO:182), a first gene encoding a KO polypeptide (SEQ ID NO:171, SEQ ID NO:172), a second gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a CPR polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:195, SEQ ID NO:196), a gene encoding a C. maxima GA20ox-1 polypeptide (SEQ ID NO:39, SEQ ID NO:40), a gene encoding a G. fujikuroi P450-3-4 polypeptide (SEQ ID NO:185, SEQ ID NO:186), a gene encoding a G. fujikuroi DES-1 polypeptide (SEQ ID NO:25, SEQ ID NO:26), and a gene encoding an A. niger CPR16 polypeptide (SEQ ID NO:157, SEQ ID NO:158) accumulates gibberellins. See
In some embodiments, expression of a gene encoding a KAO polypeptide (such as, but not limited to, a KAO11 polypeptide having the amino acid sequence SEQ ID NO:64) in an ent-kaurenoic acid-producing S. cerevisiae strain that further coexpresses C. maxima GA20ox (SEQ ID NO:39, SEQ ID NO:40) and Oryza sativa GA13ox (SEQ ID NO:97, SEQ ID NO:98) results in accumulation of GA9 and GA20. See
In some embodiments, an S. cerevisiae strain (strain “P”) comprising a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179, SEQ ID NO:180), a gene encoding a KS polypeptide (SEQ ID NO:181, SEQ ID NO:182), a first gene encoding a KO polypeptide (SEQ ID NO:171, SEQ ID NO:172), a second gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a CPR polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:195, SEQ ID NO:196), a gene encoding a P. sativum KAO11 polypeptide (SEQ ID NO:63, SEQ ID NO:64), a gene encoding a C. maxima GA7ox polypeptide (SEQ ID NO:151, SEQ ID NO:152), a gene encoding a B. diazoefficiens ADH polypeptide (SEQ ID NO:115, SEQ ID NO:116), a gene encoding a B. diazoefficiens CYP112 polypeptide (SEQ ID NO:123, SEQ ID NO:124), a gene encoding a P. putida ferredoxin polypeptide (SEQ ID NO:147, SEQ ID NO:148), and a gene encoding a P. putida ferredoxin reductase polypeptide (SEQ ID NO:149, SEQ ID NO:150) accumulates GA9. See Example 7,
In some embodiments, an S. cerevisiae strain (strain “U”) comprising a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179, SEQ ID NO:180), a gene encoding a KS polypeptide (SEQ ID NO:181, SEQ ID NO:182), a first gene encoding a KO polypeptide (SEQ ID NO:171, SEQ ID NO:172), a second gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a CPR polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:195, SEQ ID NO:196), a gene encoding an S. manihoticola KAO4 polypeptide (SEQ ID NO:73, SEQ ID NO:74), a gene encoding a KO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a B. diazoefficiens ADH polypeptide (SEQ ID NO:115, SEQ ID NO:116), a gene encoding a B. diazoefficiens CYP112 polypeptide (SEQ ID NO:123, SEQ ID NO:124), a gene encoding a P. putida ferredoxin polypeptide (SEQ ID NO:147, SEQ ID NO:148), and a gene encoding a P. putida ferredoxin reductase polypeptide (SEQ ID NO:149, SEQ ID NO:150) accumulates GA4. See Example 7,
In some embodiments, an S. cerevisiae strain comprising a gene encoding a DAP1-2 polypeptide (SEQ ID NO:212, SEQ ID NO:213), a gene encoding a CytB5-2 polypeptide (SEQ ID NO:238, SEQ ID NO:239), a gene encoding a CytB5red-4 polypeptide (SEQ ID NO:240, SEQ ID NO:241), a gene encoding a FfCytB5-1 polypeptide (SEQ ID NO:159, SEQ ID NO:160), a gene encoding a FfCytB5red-1 polypeptide (SEQ ID NO:01, SEQ ID NO:02), a gene encoding an KAO11 polypeptide (SEQ ID NO:63, SEQ ID NO:64), a gene encoding CPR12 polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding a CDPS-KS6 polypeptide (SEQ ID NO:101, SEQ ID NO:102), a gene encoding a KS5 polypeptide (SEQ ID NO:181, SEQ ID NO:182), a gene encoding a GGPPS-7 polypeptide (SEQ ID NO:177, SEQ ID NO:178), a gene encoding a KO1 polypeptide (SEQ ID NO:171, SEQ ID NO:172), a gene encoding a O. sativa GA13ox-1 polypeptide (SEQ ID NO:97, SEQ ID NO:98) a gene encoding a C. maxima GA20ox-4 polypeptide (SEQ ID NO:39, SEQ ID NO:40), and a gene encoding a M. macrocarpus GA3ox-1 polypeptide (SEQ ID NO:27, SEQ ID NO:28). The strain produces GA4 and other gibberellin intermediates. See Example 12,
In some embodiments, an S. cerevisiae strain comprising a gene encoding a DAP1-2 polypeptide (SEQ ID NO:212, SEQ ID NO:213), a gene encoding an ICE2-2 polypeptide (SEQ ID NO:206, SEQ ID NO:206), a gene encoding a CDPS-KS6 polypeptide (SEQ ID NO:101, SEQ ID NO:102), a gene encoding a KS5 polypeptide (SEQ ID NO:181, SEQ ID NO:182), a gene encoding a FfCytB5-1 polypeptide (SEQ ID NO:159, SEQ ID NO:160) a gene encoding a FfCytB5red-1 polypeptide (SEQ ID NO:01, SEQ ID NO:02), a gene encoding an KAO3 polypeptide (SEQ ID NO:145, SEQ ID NO:146), a gene encoding a CPR19 polypeptide (SEQ ID NO:193, SEQ ID NO:194), a gene encoding CPR12 polypeptide (SEQ ID NO:167, SEQ ID NO:168), a gene encoding a RsKO polypeptide (SEQ ID NO:169, SEQ ID NO:170), a gene encoding a GGPPS-7 polypeptide (SEQ ID NO:177, SEQ ID NO:178), a gene encoding a KO1 polypeptide (SEQ ID NO:171, SEQ ID NO:172), a gene encoding a P450-2-1 polypeptide (SEQ ID NO:79, SEQ ID NO:80) a gene encoding a KAO4 polypeptide (SEQ ID NO:73, SEQ ID NO:74), and a gene encoding a DES-1 polypeptide (SEQ ID NO:25, SEQ ID NO:26). The strain produces GA3 and other gibberellin intermediates. See Example 11 and Tables 19 and 20.
In some aspects, a gibberellin-producing host or gibberellin precursor-producing host comprises a damage resistance protein 1 (DAP1) polypeptide. In some embodiments, the DAP1 polypeptide is a DAP1 polypeptide as set forth in GenBank Accession No. YPL170W (SEQ ID NO:223, SEQ ID NO:224). In some aspects, the DAP1 enzyme is a G. fujikuroi DAP1 polypeptide is a polypeptide having the amino acid sequence set forth in SEQ ID NO:215, SEQ ID NO:217, or SEQ ID NO:219 (encoded by a nucleotide sequence set forth in SEQ ID NO:214, SEQ ID NO:216, or SEQ ID NO:217, respectively). In some aspects, expression of a DAP polypeptide increases cytochrome P450 activity.
In some aspects, a gibberellin-producing host or gibberellin precursor-producing host comprises inheritance of cortical ER protein 2 (ICE2) polypeptide. In some aspects, the ICE2 polypeptide can be a G. fujikuroi ICE2 (SEQ ID NO:205, SEQ ID NO:206). In some aspects, ICE2 is overexpressed.
In some embodiments, one or more endogenous genes encoding one or more alcohol dehydrogenase polypeptides are disrupted in a host. In some aspects, an alcohol dehydrogenase is knocked out or disrupted individually or in combination with one or more additional alcohol dehydrogenases. In some aspects, disruption of an endogenous alcohol dehydrogenase prevents reduction of aldehyde pathway intermediates to their corresponding alcohols. For example, disruption of one or more alcohol dehydrogeases can prevent reduction of GA12-aldehyde, GA14-aldehyde, kaurenal, GA24, and/or GA36. In some aspects, disruption of an endogenous alcohol dehydrogenase results in an increased accumulation of gibberellins.
Gibberellin production can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, LC-MS, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraviolet visible spectroscopy/spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR). In some aspects, GA3 accumulates at least 100 mg/liter in fed batch fermentation methods.
Functional homologs of the polypeptides described above are also suitable for use in producing gibberellins in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides (“domain swapping”). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term “functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of gibberellin biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using a cytochrome P450 monooxygenase, cytochrome P450 reductase, and/or 2-ODD amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a gibberellin biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in gibberellin biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
Conserved regions can be identified by locating a region within the primary amino acid sequence of a gibberellin biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
For example, polypeptides suitable for producing gibberellins in a recombinant host include functional homologs of cytochrome P450 monooxygenase, cytochrome P450 reductase, and/or 2-ODD. Methods to modify the substrate specificity of, for example, cytochrome P450 monooxygenase, cytochrome P450 reductase, and/or 2-ODD, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example, see Osmani et al., 2009, Phytochemistry 70: 325-47.
A candidate sequence typically has a length that is from 80% to 200% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120% of the length of the reference sequence, or any range between. A percent (%) identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or the amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment).
ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: % age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: % age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
To determine percent (%) identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the % identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
The term “% identity” as used herein about amino acid sequences means the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
[(identical amino acid residues)/(Length of alignment−total number of gaps in alignment)]×100
The protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:
Furthermore the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly the program calculates the identity only for these matching segments. Therefore the identity calculated in this way is referred to as local identity.
It will be appreciated that functional cytochrome P450, cytochrome P450 monooxygenase, cytochrome P450 reductase, and/or 2-ODD proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. In some embodiments, cytochrome P450, cytochrome P450 monooxygenase, cytochrome P450 reductase, and/or 2-ODD proteins are fusion proteins. The terms “chimera,” “fusion polypeptide,” “fusion protein,” “fusion enzyme,” “fusion construct,” “chimeric protein,” “chimeric polypeptide,” “chimeric construct,” and “chimeric enzyme” can be used interchangeably herein to refer to polypeptides engineered through the joining of two or more genes that code for different polypeptides (i.e., a polypeptide operatively-linked to a different polypeptide). For example, a polypeptide encoded by a nucleic acid sequence containing a coding sequence from one nucleic acid molecule and the coding sequence from another nucleic acid molecule in which the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated in a host cell, the protein is produced containing the two proteins. The two molecules can be adjacent in the construct or separated by a linker polypeptide that contains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide. A chimeric or fusion protein provided herein can include one or more For example, a non-limiting example of a fusion protein can include a CDPS gene fused to a KS gene to generate a CDPS-KS fusion protein when expressed. In some embodiments, a nucleic acid sequence encoding a cytochrome P450, cytochrome P450 monooxygenase, cytochrome P450 reductase, and/or 2-ODD polypeptide can include a tag sequence that encodes a “tag” designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag™ tag (Kodak, New Haven, Conn.). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term “domain swapping” is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein.
In some embodiments, a protein is a protein altered by circular permutation, which consists in the covalent attachment of the ends of a protein that would be opened elsewhere afterwards. Thus, the order of the sequence is altered without causing changes in the amino acids of the protein. In some embodiments, a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct. In some embodiments, a polypeptide disclosed herein is altered by circular permutation.
A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism. A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids may be introduced at positions other than the position where the native sequence is found or kept extrachromosomally in episomes.
As used herein, the term “regulatory region” refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
One or more genes can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of gibberellin precursor and/or gibberellin production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. For example, a gibberellin biosynthesis gene cluster, or a UGT gene cluster, can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species. As another example, a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for gibberellin precursor or gibberellin production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates towards gibberellin precursor or gibberellin biosynthesis. For example, it may be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase gibberellin precursor or gibberellin production, e.g., by downregulating squalene epoxidase. As another example, it may be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein. In such cases, a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.
Recombinant hosts can be used to express polypeptides for the producing gibberellins, including mammalian, insect, plant, and algal cells. A number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast, and fungi. A species and strain selected for use as a gibberellin production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
In some embodiments, the bacterial cell comprises Escherichia cells, Lactobacillus cells, Lactococcus cells, Corynebacterium cells, Acetobacter cells, Acinetobacter cells, Pseudomonas cells, or Streptomyces cells.
In some embodiments, the fungal cell comprises a yeast cell. For example, the yeast cell can be a Saccharomycete. The yeast cell can comprise a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species. In an embodiment, the yeast cell is a cell from the Saccharomyces cerevisiae species. In another embodiment, the fungal cell of the fungal cell comprises a filamentous fungal cell.
Typically, the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate the production of a gibberellin precursor and/or gibberellin compound. For example, the period of time can be approximately 120 hours. Growth in a fermenter can be performed with agitation. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture. Depending on the particular microorganism used in the method, other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, ent-kaurene and ent-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
As used herein “a carbon source” or “carbon sources” can include any molecule that can be metabolized by a recombinant host cell to facilitate growth and/or production of the gibberellins. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose, maltodextrin, mannitol, other sugars or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate the production of a gibberellin precursor and/or gibberellin compound, the gibberellin precursor and/or gibberellin compound can then be recovered from the culture using various techniques known in the art. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C-18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See for example, WO 2009/140394.
It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate gibberellin precursors and/or gibberellins.
Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g., ent-kaurenoic acid, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, GA3. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable. For example, suitable species can be in a genus such as Agaricus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Bradyrhizobium, Rhizobium, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces or Yarrowia. Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Bradyrhizobium japonicum, Xanthophyllomyces dendrorhous, F. fujikuroi/G. fujikuroi, Candida utilis, Candida glabrata, Candida albicans, and Yarrowia lipolytica.
In some embodiments, a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Corynebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
In some embodiments, a microorganism can be an Ascomycete such as G. fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, A. niger, Yarrowia lipolytica, Ashbya gossypii, or S. cerevisiae.
In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
In some embodiments, a microorganism can be a cyanobacterial cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis.
Saccharomyces is a widely used organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms.
Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing gibberellins. E. coli
E. coli, another widely-used organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, the terpene precursors for producing large amounts of gibberellins are already produced by endogenous genes. Thus, modules comprising recombinant genes for gibberellin biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
Arxula adeninivorans (Blastobotrys adeninivorans)
Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42° C., above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Yarrowia lipolytica
Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g. alkanes, fatty acids, oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorganism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biochimie 91(6):692-6; Bankar et al., 2009, Appl Microbiol Biotechnol. 84(5):847-65.
Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 2011, Process Biochemistry 46(1):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41:312-7).
Rhodosporidium toruloides
Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid-production pathways. See, e.g., Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al., 2011, Applied Microbiology and Biotechnology 90(4):1219-27).
Candida boidinii
Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., Mattanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha (Pichia angusta)
Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
Kluyveromyces lactis
Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al., 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris
Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen et al., 2014, N Biotechnol. 31(6):532-7.
Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
It can be appreciated that the recombinant host cell disclosed herein can comprise a plant cell, a mammalian cell, an insect cell, a fungal cell, comprising a yeast cell, wherein the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species or is a Saccharomycete or is a Saccharomyces cerevisiae cell, an algal cell or a bacterial cell, comprising Escherichia cells, Lactobacillus cells, Lactococcus cells, Cornebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
Various plants can be used as recombinant host cells (e.g., plant cells, both monocotyledenous and dicotyledenous). In an embodiment, the plants or host cells used in the methods can be derived from monocots, particularly the members of the taxonomic family known as the Gramineae. This includes all members of the grass family of which the edible varieties are known as cereals. The cereals include a wide variety of species such as wheat (Triticum sps.), rice (Oryza sps.) barley (Hordeum sps.) oats, (Avena sps.) rye (Secale sps.), corn (maize) [Zea sps.) and millet (Pennisettum sps.). In another embodiment, the plants or host cells used can be derived from dicots (e.g., soybean (Glycine spp.)). In order to produce transgenic plants that produce gibberellins, plant cells or tissues derived from them are transformed or integrated with genes coding for various enzymes the result in the production of gibberellins. The transgenic plant cells are cultured in medium containing the appropriate selection agent to identify and select for plant cells which express the heterologous nucleic acid sequence. After plant cells that express the heterologous nucleic acid sequence are selected, whole plants can be regenerated from the selected transgenic plant cells. Techniques for regenerating whole plants from transformed plant cells are generally known in the art.
Plant cells or tissues can be transformed with expression constructs (i.e., heterologous nucleic acid constructs) using a variety of standard techniques. In some embodiments, the heterologous nucleic acid sequences can be stably integrated into the host cell genome so that the integrated nucleic acid sequences are passed onto successive plant generations. The skilled artisan will recognize that a wide variety of transformation techniques exist in the art. Any technique that is suitable for the target host plant may be employed. For example, the nucleic acid sequences can be introduced in a variety of forms including, but not limited to, as a strand of DNA, in a plasmid, or in an artificial chromosome. The introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to calcium-phosphate-DNA co-precipitation, electroporation, microinjection, Agrobacterium-mediated transformation, liposome-mediated transformation, protoplast fusion or microprojectile bombardment. When Agrobacterium is used for plant cell transformation, a vector is introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and gall. In some embodiments, Agrobacterium can be used as the vehicle for transforming host plant cells. The expression or transcription construct bordered by the T-DNA border region(s) is inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, for example pRK2 or derivatives thereof. Alternatively, one may insert the sequences to be expressed in plant cells into a vector containing separate replication sequences, one of which stabilizes the vector in E. coli, and the other in Agrobacterium. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like.
It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce gibberellin precursors and/or gibberellins. For example, a first microorganism can comprise one or more biosynthesis genes for producing a gibberellin precursor, while a second microorganism comprises gibberellin biosynthesis genes. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Alternatively, the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., ent-kaurenoic acid, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as GA3. The product produced by the second, or final microorganism is then recovered.
A number of different methods can be used to isolate and purify the gibberellin precursors and/or gibberellin compounds produced by the methods and host cells disclosed herein. For example, the isolating steps may comprise: (a) contacting the cell culture comprising the gibberellin precursor and/or the gibberellin compound with: (i) one or more adsorbent resins in a packed column in order to bind at least a portion of the gibberellin precursor and/or the gibberellin compound to the resin, thereby isolating the gibberellin precursor and/or the gibberellin compound; or (ii) one or more ion exchange or reversed-phase chromatography columns in order to bind at least a portion of the gibberellin precursor and/or the gibberellin compound in the column, thereby isolating the gibberellin precursor and/or the gibberellin compound; or (b) crystallizing and/or extracting the gibberellin precursor and/or the gibberellin compound from the cell culture, thereby isolating the gibberellin precursor and/or the gibberellin compound; or (c) separating the cell culture into a solid phase and a liquid phase, wherein the liquid phase comprises the gibberellin precursor and/or the gibberellin compound; and (i) contacting the liquid phase with one or more adsorbent resins in order to bind at least a portion of the gibberellin precursor and/or the gibberellin compound to the resin, thereby isolating the gibberellin precursor and/or the gibberellin compound; (ii) contacting the liquid phase with one or more ion exchange or reversed-phase chromatography columns in order to bind at least a portion of the gibberellin precursor and/or the gibberellin compound in the column, thereby isolating the gibberellin precursor and/or the gibberellin compound; or (iii) crystallizing and/or extracting the gibberellin precursor and/or the gibberellin compound from the liquid phase, thereby isolating the gibberellin precursor and/or the gibberellin compound.
In an embodiment, the isolating step can comprise, separating the solid phase from the liquid phase using a process comprising tangential flow filtration with diafiltration membranes to generate a permeate stream comprising the gibberellin precursor and/or the gibberellin compound, wherein the membranes used in the tangential flow filtration are ultrafiltration or nanofiltration membranes. In an embodiment, the permeate stream is extracted by an organic solvent which phase-separates from the aqueous phase to generate an extracted gibberellin product in the organic solvent
Optionally the permeate stream containing the gibberellin product could be concentrated by some combination of reverse osmosis, nanofiltration, and evaporation to produce a crystallized gibberellin precursor and/or the gibberellin compound.
The aqueous gibberellin-containing permeate or the concentrate can be extracted by an organic solvent which phase-separates from the aqueous phase. The pH of the aqueous phase is adjusted to less than 4.0, or less than 3.0, in order to protonate the gibberellin molecules and ensure they partition into the organic phase to a high degree. The solvent extraction could be performed in a counter-current extraction centrifuge such as a Podbelniak extractor, or in a counter-current extraction column such as a Karr or Scheibel column. This yields the gibberellin product in an organic solvent suitable for subsequent purification processing.
It will be understood that organic solvent extraction can be replaced with a series of process operations which yield a similar organic solution of gibberellins. The series of process operations would include (a) precipitation of gibberellins from the aqueous concentrate produced by addition of acid until pH is less than 4.0 or less than 3.0; (b) filtration and optionally water-washing of the resulting gibberellins-containing solids; and (c) dissolution of the filtered gibberellins-containing solids into an organic solvent suitable for purification processing.
Optionally the organic extract can be contacted with carbon to adsorb impurities and color bodies. Optionally the carbon contacting can be done by mixing carbon in the organic extract and filtering the carbon out of the resulting suspension, or by feeding the organic extract to a column or filter containing a fixed bed of carbon and collecting a purified effluent stream. The organic extract can be crystallized by concentrating the solution evaporatively. The resulting gibberellins product crystals can be filtered, washed, and dried to yield a high-purity gibberellins product.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Liquid chromatography-mass spectrometry (LC-MS) analyses were performed on Waters ACQUITY UPLC® (Waters Corporation) with a Waters ACQUITY UPLC® BEH C18 column (2.1×50 mm, 1.7 μm particles, 130 Å pore size) equipped with a pre-column (2.1×5 mm, 1.7 μm particles, 130 Å pore size) coupled to a Waters ACQUITY TQD triple quadropole mass spectrometer with electrospray ionization (ESI) operated in negative ionization mode. Compound separation was achieved using a gradient of the two mobile phases: phase A (water with 0.1% formic acid) and phase B (MeCN with 0.1% formic acid) were separated by increasing from 20% to 50% B between 0.3 to 2.0 minutes, increasing to 100% B at 2.01 minutes and holding 100% B for 0.6 minutes, and re-equilibrating for 0.6 minutes. The flow rate was 0.6 mL/min, and the column temperature was set at 55° C. Gibberellins were monitored using SIM (Single Ion Monitoring) and quantified by comparing against authentic standards.
An ent-kaurenoic acid-producing S. cerevisiae strain comprising genes encoding a truncated copalyl diphosphate synthase (CDPS) polypeptide (SEQ ID NO:179 (nt), SEQ ID NO:180 (aa)), a kaurene synthase (KS) polypeptide (SEQ ID NO:181 (nt), SEQ ID NO:182 (aa)), a first KO polypeptide (SEQ ID NO:171 (nt), SEQ ID NO:172 (aa)), a second KO polypeptide (SEQ ID NO:169 (nt), SEQ ID NO:170 (aa)), a CPR polypeptide (SEQ ID NO:167 (nt), SEQ ID NO:168 (aa)), and an ERG20-GGPPS7 polypeptide (SEQ ID NO:195 (nt), SEQ ID NO:196 (aa)) was engineered to accumulate gibberellins. Strains “A,” “N,” and “F” were transformed into this ent-kaurenoic acid-producing strain background; the genes of Table 2 or Table 3 were introduced into the strain using the USER™ based yeast integration vector system. See, e.g., Mikkelsen et al., 2012, Metabolic Engineering 14:104-11. See also, the pathway described in
G. fujikuroi
G. fujikuroi
S. manihoticola
G. fujikuroi
G. fujikuroi
G. fujikuroi
G. fujikuroi
G. fujikuroi
S. manihoticola
G. fujikuroi
A. niger
Furthermore, the ent-kaurenoic acid-producing S. cerevisiae strain described above was also transformed with the genes of Table 4 using the USER™ cloning based yeast integration system to engineer strain “F.” See the pathway described in
C. maxima
G. fujikuroi
S. manihoticola
G. fujikuroi
A. niger
Gibberellin accumulation was observed with these recombinant S. cerevisiae strains and was measured using one of two LC-MS methods. In the first method, LC-MS analysis was performed using a Waters ACQUITY I-class UPLC system fitted with a Waters ACQUITY UPLC® BEH shield RP18 column (2.1×50 mm, 1.7 μm particles, 130 Å pore size) equipped with an ACQUITY UPLC® BEH C18 VanGuard pre-column (130 Å, 1.7 μm, 2.1 mm×5 mm) connected to a Waters Xevo SQ Detector 2 single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. Compound separation was carried out using mobile phase of eluent B (ACN with 0.1% formic acid) and eluent A (water with 0.1% formic acid) using gradient separation. Quantification of gibberellins was performed by comparing obtained signals with authentic standards. Gibberellin accumulation was detected using single ion reaction (SIR) in negative ionization mode using the traces described in Table 5. In the second method, LC-MS analysis was performed using a Waters ACQUITY I-class UPLC system fitted with a Waters ACQUITY UPLC® BEH shield RP18 column (2.1×50 mm, 1.7 μm particles, 130 Å pore size) equipped with an ACQUITY UPLC® BEH C18 VanGuard pre-column (130 Å, 1.7 μm, 2.1 mm×5 mm) connected to a Waters XEVO® G2-S quadrupole time-of-flight (QTOF) mass spectrometer equipped with an electrospray ionization (ESI) source operated in negative ionization mode. Compound separation was carried out using the gradient of the first LC-MS method. Gibberellin accumulation was detected by investigating extracted ion chromatograms (EICs) corresponding to their theoretical accurate mass.
As shown in
As shown in
The expression of GGPPS producing genes alone has been shown to cause cell toxicity, therefore, GGPPS was removed by the expression of CDPS and KS genes. CDPS-KS bifunctional genes were constructed to determine the efficiency of each CDPS/KS combination for removing GGPPS by converting GGPPS to kaurenoic acid. CDPS-KS bifunctional fusion genes were comparatively tested in a yeast strain inserted with CytB5-1 and CytB5red-1. The strain was then transformed with CPR12 (SEQ ID NO:167 (nt) and SEQ ID NO:168 (aa)), RsKO_GA (SEQ ID NO:169 (nt) and SEQ ID NO:170 (aa)), GGPPS7 (SEQ ID NO:176 (aa) and SEQ ID NO:178 (aa)), KO1 (SEQ ID NO:171 (nt) and SEQ ID NO:172 (aa)), and either CDPS-KS6+KS5 (SEQ ID NO:101 (nt) and SEQ ID NO:102 (aa), and SEQ ID NO:181 (nt) and SEQ ID NO:182 (aa)), CDPS-KS6 (SEQ ID NO:101 (aa) and SEQ ID NO:102 (nt)), CDPS-KS4 (SEQ ID NO:226 (nt) and SEQ ID NO:227 (aa)), or CDPS-KS9 (SEQ ID NO:228 (nt) and SEQ ID NO:229 (aa)). The expression of the giberellin pathway genes along with CDPS-KS bifunctional genes were tested to determine the production level of kaurenoic acid. Greater levels of production of kaurenoic acid by a bifunctional CDPS-KS gene alone were produced by the expression of the CDPS-KS6 gene (115.14 μM) and this was enhanced by the co-expression of KS5 (CDPS-KS6+KS5) (182.70 μM). The bifunctional CDPS-K4 was less effective in the removal of GGPP as evidenced by the smaller amount of production of kaurenoic acid (8.80 μM) when compared to bifunctional CDPS-KS6 (see Table 6).
The production level of gibberellins and gibberellin metabolites can vary depending on the expression of a KAO gene. To determine the amount of GA12 and GA14 produced by KAO activity, a yeast strain containing KO1 (SEQ ID NO:171 (nt) and SEQ ID NO:172 (aa)) and CPR19 (SEQ ID NO:193 (nt) and SEQ ID NO:194 (aa)) was transformed with CDPS-KS6 (SEQ ID NO:101), KS5 (SEQ ID NO:181), GGPPS7 (SEQ ID NO:177), KO1 (SEQ ID NO:171), KAO and CPR genes using USER™ based DNA assembler vectors and NatMx marker. Transformants were then grown and metabolites were analyzed using LC-MS. Yeast strains co-expressed KAO3/CPR19 genes (SEQ ID NO:230 and SEQ ID NO:193), KAO4/CPR17 (SEQ ID NO:73 and SEQ ID NO:187) or CPR19 (SEQ ID NO:193) genes, or KAO5/CPR12 (SEQ ID NO:61 and SEQ ID NO:167) or CPR19 genes (SEQ ID NO:193). The KAO3 and KAO5 genes used were obtained from Integrated DNA Technologies (IDT), and the KAO4 gene used was obtained from GeneArt™ (Invitrogen). Expression of KAO3 resulted in the production 1205 (AUC) of GA12 and 25055 (AUC) of GA14. Expression of KAO4 resulted in the production 4175 (AUC) GA12 and 127115 (AUC) GA14. Lastly, expression of KAO5 resulted in the production of 1605 (AUC) GA14.
Additional yeast studies were conducted to determine the production of gibberellins by various codon-optimized versions of KAO. KAO1 and KAO3 were both codon-optimized versions of F. fujikuroi while KAO2 and KAO4 were codon-optimized versions of F. proliferatum and S. manihoticola, respectively. A yeast train containing FfCytB5-1 (SEQ ID NO:159 (nt) and SEQ ID NO:160 (aa)), FfCytB5red-1 (SEQ ID NO:01 (nt) and SEQ ID NO:02 (aa)), CPR19 (SEQ ID NO:193 (nt) and SEQ ID NO:194 (aa)), RsKO-GA (SEQ ID NO:169 (nt) and SEQ ID NO:170 (aa)), KS5 (SEQ ID NO:181 (nt) and SEQ ID NO:182 (aa)), tCDPS5 (SEQ ID NO:179 (nt) and SEQ ID NO:180 (aa)), GGPPS7 (SEQ ID NO:177 (nt) and SEQ ID NO:178 (aa)), and KO1 (SEQ ID NO:171 (nt) and SEQ ID NO:172 (aa)) was transformed with P450-3-1 (SEQ ID NO:45), P450-2-4 (SEQ ID NO:141), P450-3-4 (SEQ ID NO:185), DES-1 (SEQ ID NO:25), and either KAO1 (SEQ ID NO:89), KAO3 (SEQ ID NO:145), KAO4 (SEQ ID NO:73) or KAO5 (SEQ ID NO:61). USER™ based DNA assembler vectors and URA3 markers were used. Transformants were then grown and metabolites were analyzed using LC-MS. Various amounts of metabolites from GA14 and further downstream the gibberellin pathway were produced (see Table 8). All numerical values in Tables 7 and 8 are area under curve (AUC).
Gibberellin acid 14 (GA14) is converted to GA4 and GA1 by P450 enzymes. A comparative study of P450-2 homologs was conducted to determine the production level of gibberellins. A yeast strain inserted with P450-3-4 (SEQ ID NO:141 (nt) and SEQ ID NO:142 (aa)), KO1 (SEQ ID NO:170 (nt) and SEQ ID NO:171 (aa)), GGPPS7 (SEQ ID NO:177 (nt) and SEQ ID NO:178 (aa)), CDPS-KS6 (SEQ ID NO:101 (nt) and SEQ ID NO:102 (aa)), KAO4 (SEQ ID NO:73 (nt) and SEQ ID NO:74 (aa)), FfCytB5-1 (SEQ ID NO:159 (nt) and SEQ ID NO:160 (aa)), CPR1 (SEQ ID NO:165 (nt) and SEQ ID NO:166 (aa)), CPR19 (SEQ ID NO:193 (nt) and SEQ ID NO:194 (aa)), and various P450-2 genes. To identify which P450-2 gene was more efficient at the production of GA1, P450-2-1 (SEQ ID NO:79 (nt) and SEQ ID NO:80 (aa)), P450-2-8 (SEQ ID NO:232 (nt) and SEQ ID NO:233 (aa)), P450-2-9 (SEQ ID NO:234 (nt) and SEQ ID NO:235 (aa)), and P450-2-10 (SEQ ID NO:236 (nt) and SEQ ID NO:237 (aa)) were tested. The combination of genes resulted in the production of GA1. P450-2-1 produced greater levels of both GA1 (30309 AUC) and GA4 (34370 AUC) when compared to the other P450-2 enzymes tested, while P450-2-10 produced a smaller amount of GA1 (13611 AUC) and P450-2-8 produced a smaller amount of GA4 (17854 AUC) when compared to the other P450-2 enzymes tested (see Table 9).
P450-2 enzymes use GA14 as a substrate to produce GA4. To determine the production level of GA4 by P450-2 activity, P450-2 genes were introduced into a GA14 producing strain by integration into the yeast genome using a USER™ cloning based vector system. Each P450 gene was introduced using the URA3 selection marker. P450-2-1 and P450-2-6 (SEQ ID NO:17, SEQ ID NO:18) produced surprising levels of GA4 that were greater levels of GA4 (581,138 AUC and 279,002 AUC, respectively) when compared to the other P450-2 enzymes tested, while P450-2-4 produced a smaller amount of GA4 (3456.88 AUC) (see Table 10). All numerical values in Tables 9 and 10 are area under the curve (AUC).
Fusarium fujikuroi P450-2-1
Fusarium fujikuroi P450-2-4
Ustilaginoidea virens P450-2-5
Fusarium oxysporum P450-2-6
Using the USER™ cloning based yeast integration system, the genes in Table 11 were individually introduced into an S. cerevisiae strain that further comprised a gene encoding a G. fujikuroi CPR5 polypeptide (SEQ ID NO:47 (nt), SEQ ID NO:48 (aa)), a gene encoding a CPR12 polypeptide (SEQ ID NO:167 (nt), SEQ ID NO:168 (aa)), a gene encoding an A. thaliana KS5 polypeptide (SEQ ID NO:181 (nt), SEQ ID NO:182 (aa)), a gene encoding a truncated Zea mays CDPS polypeptide (SEQ ID NO:179 (nt), SEQ ID NO:180 (aa)), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:220 (nt), SEQ ID NO:221 (aa)), and a gene encoding a Stevia rebaudiana KO1 polypeptide (SEQ ID NO:171 (nt), SEQ ID NO:172 (aa)). See the pathway described in
A. thaliana KAO5
A. thaliana KAO6
H. vulgare KAO9
P. sativum KAO10
P. sativum KAO11
S. manihoticola KAO4
Co-expression of C. maxima GA20ox-4 (SEQ ID NO:39 (nt), SEQ ID NO:40 (aa)), Oryza sativa GA13ox (SEQ ID NO:97, SEQ ID NO:98), P. sativum KAO11 (SEQ ID NO:63 (nt), SEQ ID NO:64 (aa)), and C. maxima Ga7ox-1 (SEQ ID NO:151 (nt), SEQ ID NO:152 (aa)) in the kaurenoic acid-producing S. cerevisiae strain further resulted in accumulation of GA9 and GA20. See the pathway described in
Using the USER™ cloning based yeast integration system, the genes in Table 12 or Table 13 were introduced into an S. cerevisiae strain that further comprised a gene encoding a G. fujikuroi CPR5 polypeptide (SEQ ID NO:47 (nt), SEQ ID NO:48 (aa)), a gene encoding a CPR12 polypeptide (SEQ ID NO:167 (nt), SEQ ID NO:168 (aa)), a gene encoding an A. thaliana KS5 polypeptide (SEQ ID NO:181 (nt), SEQ ID NO:182 (aa)), a gene encoding a truncated Z. mays CDPS polypeptide (SEQ ID NO:179 (nt), SEQ ID NO:180 (aa)), a gene encoding an ERG20-GGPPS7 polypeptide (SEQ ID NO:220 (nt), SEQ ID NO:221 (aa)), and a gene encoding a Stevia rebaudiana KO1 polypeptide (SEQ ID NO:171 (nt), SEQ ID NO:172 (aa)). See the pathways described in
P. sativum
C. maxima
B. diazoefficiens
B. diazoefficiens
P. putida
P. putida
S. manihoticola
B. diazoefficiens
B. diazoefficiens
P. putida
P. putida
An S. cerevisiae strain comprising a gene encoding a P450-1 polypeptide (SEQ ID NO:87 (nt), SEQ ID NO:88 (aa)) or a P450-1 polypeptide (SEQ ID NO:145 (nt), SEQ ID NO:146 (aa)), a KAO4 polypeptide (SEQ ID NO:73 (nt), SEQ ID NO:74 (aa)), or a KAO1 polypeptide (SEQ ID NO:89 (nt), SEQ ID NO:90 (aa)) was engineered to accumulate kaurenoic acid, as described in Example 2. Using the USER™ based yeast integration vector system, S. manihoticola KAO4 polypeptide (SEQ ID NO:73 (nt), SEQ ID NO:74 (aa)) or G. fujikuroi KAO1 polypeptide (SEQ ID NO:89 (nt), SEQ ID NO:90 (aa)) individually introduced into the S. cerevisiae strain. As shown in
Using the USER™ based yeast integration vector system, the genes in Table 14, Table 15, or Table 16 were introduced into an S. cerevisiae strain that further comprised a gene encoding a truncated CDPS polypeptide (SEQ ID NO:179 (nt), SEQ ID NO:180 (aa)), a KS polypeptide (SEQ ID NO:181 (nt), SEQ ID NO:182 (aa)), a KO polypeptide (SEQ ID NO:171 (nt), SEQ ID NO:172 (aa)), a CPR polypeptide (SEQ ID NO:167 (nt), SEQ ID NO:168 (aa)), and an ERG20-GGPPS7 polypeptide (SEQ ID NO:195 (nt), SEQ ID NO:196 (aa)). The strains described in Tables 14-16 were identical, except that they comprised either CPR14, CPR15, or CPR16.
G. fujikuroi
G. fujikuroi
A. niger
G. fujikuroi
G. fujikuroi
G. fujikuroi
G. fujikuroi
G. fujikuroi
Phaeosphaeria sp.
G. fujikuroi
G. fujikuroi
G. fujikuroi
G. fujikuroi
G. fujikuroi
Candida
G. fujikuroi
apicola
G. fujikuroi
G. fujikuroi
As shown in
Using the USER™ based yeast integration vector system, the genes in Table 17 were stably integrated into an S. cerevisiae strain. The strain was grown in a 2 L Sartorius fermentor using a fed batch process. Temperature, pH, agitation, and aeration rate were controlled throughout the cultivation. The temperature was maintained at 30° C. Air was used for sparging the bioreactor at 1 vvm (L gas/(L liquid×min)). pH was controlled at pH 5.0 by automatic addition of NH4OH. An 8% NH4OH solution was used for the first 45 hours of the process; a 16% solution was used for the final part. The stirrer speed was initially set to 800 rpm and increased to up to 1600 rpm during the process. The basis for the medium used for the batch phase is 0.5 L minimal medium containing glucose, salts, vitamins and trace metals. The feed solution was either a high density glucose solution with salts, trace metals and vitamins (glucose feed) or 96% ethanol (ethanol feed). Antifoam was included in the batch medium and feed medium. The fermentation was inoculated using a seed train in shake flasks grown at 30° C. using a minimal medium with similar content as the medium used for the batch phase in the fermentation. The batch fermentation lasted for 16 hours. During the carbon-limited fed batch phase, feed was added following an exponential feed profile feeding with glucose feed from 16-70 hours and ethanol feed from 70-160 hours. Since the ethanol feed only contained the carbon source, concentrated feed components (salts, vitamins, trace metals and antifoam) were combined, sterile filtered and added to the fermentation broth once or twice per day during feeding with ethanol feed.
F. fujikuroi
F. fujikuroi
F. fujikuroi
A. thaliana
F. fujikuroi
G. fujikuroi
G. fujikuroi
R. suavissimus
R. suavissimus
Synecococcus sp.
S. rebaudiana
G. fujikuroi
As shown in
Using the USER™ based yeast integration vector system, the genes in Table 19 were stably integrated into an S. cerevisiae strain. The strain was grown in a 2 L Sartorius fermentor using a fed batch process. Temperature, pH, agitation, and aeration rate were controlled throughout the cultivation. The temperature was maintained at 30° C. Air was used for sparging the bioreactor at 1 vvm (L gas/(L liquid×min)). pH was controlled at pH 5.0 by automatic addition of NH4OH. An 8% NH4OH solution was used for the first 45 hours of the process; a 16% solution was used for the final part. The stirrer speed was initially set to 800 rpm and increased to up to 1600 rpm during the process. The basis for the medium used for the batch phase is 0.5 L minimal medium containing glucose, salts, vitamins and trace metals. The feed solution was either a high density glucose solution with salts, trace metals and vitamins (glucose feed) or 96% ethanol (ethanol feed). Antifoam was included in the batch medium and feed medium. The fermentation was inoculated using a seed train in shake flasks grown at 30° C. using a minimal medium with similar content as the medium used for the batch phase in the fermentation. The batch fermentation lasted for 16 hours. During the carbon-limited fed batch phase, feed was added following an exponential feed profile feeding with glucose feed from 16-70 hours and ethanol feed from 70-148 hours. Since the ethanol feed only contained the carbon source, concentrated feed components (salts, vitamins, trace metals and antifoam) were combined, sterile filtered and added to the fermentation broth once or twice per day during feeding with ethanol feed. After 148 hours of fermentation, the titer in growth medium was measured to be 491 mg/L (1.42 mM) of GA3 and 2.15 mM of kaurenol, 4.26 mM of kaurenal and 1.28 mM ent-kaurene. The production of GA3, GA4 and additional gibberellins and intermediate molecules at various time points during growth in the culture medium is shown in Table 20. The results demonstrate that a yeast strain comprising fungal gibberellin genes can produce gibberellins.
F. fujikuroi
F. fujikuroi
F. fujikuroi
A. thaliana
F. fujikuroi
F. fujikuroi
G. fujikuroi
G. fujikuroi
R. suavissimus
R. suavissimus
Synecococcus sp.
S. rebaudiana
G. fujikuroi
S. manihoticola
F. fujikuroi
Using the USER™ based yeast integration vector system, the genes in Table 19 were stably integrated into an S. cerevisiae strain. The strain was grown in DELFT culture medium supplemented with uracil to complement uracil auxotrophy of the strain for 96 hours. Samples were extracted with acetonitrile (80% final) and cultures were analysed using LC-MS. The production of GA4 and additional gibberellins and intermediate molecules at various time points during growth in the culture medium is shown in Table 22.
F. fujikuroi
F. fujikuroi
F. fujikuroi
P. sativum
R. suavissimus
F. fujikuroi
A. thaliana
Synecococcus sp.
S. rebaudiana
O. sativa
C. maxima
M. macrocarpus
These results demonstrated that plant GA13 ox, GA20 ox and GA3 ox genes were all active in yeast and that when combined they can catalyse the reactions from GA12 to GA53 (GA13 ox reaction) to GA9 (GA20 ox reaction) to GA20 (GA13 ox+GA20 ox reactions via either GA53 or GA9) and then further GA9 to GA4 reaction catalyzed by GA3ox genes. Further analysis revealed that sample B1 and sample C5 also contained small amounts of GA3, which thereby demonstrated a fully functional GA3 pathway from ent-kaurene based on plant derived genes (see
The “B1” strain from Example 12 was grown in a 2 L Sartorius fermentor using a fed batch process. Temperature, pH, agitation, and aeration rate were controlled throughout the cultivation. The temperature was maintained at 30° C. Air was used for sparging the bioreactor at 1 vvm (L gas/(L liquid×min)). pH was controlled at pH 5.0 by automatic addition of NH4OH. An 8% NH4OH solution was used for the first 45 hours of the process; a 16% solution was used for the final part. The stirrer speed was initially set to 800 rpm and increased to up to 1600 rpm during the process. The basis for the medium used for the batch phase is 0.5 L minimal medium containing glucose, salts, vitamins and trace metals. The feed solution was either a high density glucose solution with salts, trace metals and vitamins (glucose feed) or 96% ethanol (ethanol feed) supplemented with uracil to complement uracil auxotrophy of the strain. Antifoam was included in the batch medium and feed medium. The fermentation was inoculated using a seed train in shake flasks grown at 30° C. using a minimal medium with similar content as the medium used for the batch phase in the fermentation. The batch fermentation lasted for 16 hours. During the carbon-limited fed batch phase, feed was added following an exponential feed profile feeding with glucose feed from 16-70 hours and ethanol feed from 70-138 hours. Since the ethanol feed only contained the carbon source, concentrated feed components (salts, vitamins, trace metals and antifoam) were combined, sterile filtered and added to the fermentation broth once or twice per day during feeding with ethanol feed.
As shown in Table 23, the strain accumulated gibberellins, including, but not limited to GA3, GA4 and GA14. After approximately 138 hours of fermentation, the titer in growth medium was 1.7 μM of GA3, 73 μM of GA1, 82 μM of GA4, 1.8 μM GA7, 2400 μM of KA as well as estimated amounts of 214 μM of GA20, 1.5 μM of GA9, 134 μM of GA24, 128 μM of GA53 and 142 μM of GA12. The production of additional gibberellins and intermediate molecules at various time points during growth in the culture medium is shown in Table 23.
Using the USER™ based yeast integration vector system, the genes in Table 24 and Table 25 were stably integrated into an S. cerevisiae strain comprising the genes as shown in Table 17. The strain was grown in DELFT culture medium for 96 hours. Samples were extracted with acetonitrile (80% final) and cultures were analyzed using LC-MS. By testing plant GA13 oxidase in a GA4 producing strain, the results demonstrate that the plant GA13 oxidase can replace the fungal P450-3 enzyme, which is demonstrated by the formation of GA1 and GA3. See Table 26.
Oryza sativa
Fusarium fujikuroi
Fusarium fujikuroi
Fusarium fujikuroi
Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
This application is related to U.S. provisional patent application, Ser. No. 62/303,973, filed Mar. 4, 2016, the disclosure of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/055083 | 3/3/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62303973 | Mar 2016 | US |
