Patent Grant
11299717
This disclosure relates to production of citronellal and citronellol in recombinant hosts. In particular, this disclosure relates to the synthesis of citronellal, citronellol and citronellic acid precursors in recombinant hosts.
Citronellal is a monoterpenoid that provides a lemon scent that is commonly associated with citronella oil. In addition to providing a lemon-scented fragrance, citronellal has been shown to repel insects, including but not limited to mosquitos, and to have antifungal properties. Citronellal is also useful as a starting material for the asymmetric synthesis of related chiral compounds.
Citronellal is predominantly formed by the secondary metabolism of plants. Citronellal can be extracted from the oils of plants such as Corymbia citriodora, Cymbopogon nardus, and Cymbopogon winterianus. Citronellal is most commonly isolated by steam distillation or solvent extraction as a non-racemic mixture of its R- and S-enantiomers.
Citronellal can be reductively bioconverted to citronellol. Citronellol synthesis is usually done by hydrogenation of geraniol (trans) or nerol (cis). Similar to citronellal, citronellol is commonly used in the fragrance industry, acts as an insect repellent, and can be used as an intermediate in the synthesis of several natural terpenoids.
Because robust production of both citronellal and citronellol in, for example, the fragrance and pharmaceutical industries, significant agricultural resources in terms of land, equipment, and biomass generation are required to meet current industry needs. However, identifying alternative, highly efficient, and renewable sources of these compounds remains difficult. Moreover, greater purity of citronellal and citronellol compounds is needed but difficult to obtain from plant sources without additional processing steps. Therefore, there remains a need to develop alternative approaches for obtaining scalable amounts of highly pure citronellal and citronellol.
It is against the above background that the present invention provides certain advantages and advancements over the prior art. In particular, as set forth herein, the use of recombinant microorganisms: bacteria or yeast, to make citronellal and citronellol is disclosed.
Although this invention disclosed herein is not limited to specific advantages or functionalities the invention provides a recombinant host cell capable of producing a citronellal or a citronellic acid, comprising:
The invention also provides a recombinant host cell capable of producing a citronellal, a citronellol, or a citronellic acid, comprising:
wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cells disclosed herein:
In one aspect of the recombinant host cells disclosed herein, the GES polypeptide catalyzes the formation of geraniol from geranyl diphosphate (GPP), wherein the GPP is produced by the GPPS polypeptide converting isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from a mevalonate pathway and/or a methylerythritol 4-phosphate (MEP) pathway.
In one aspect of the recombinant host cells disclosed herein, the mevalonate pathway is an endogenous pathway or a recombinant pathway.
In one aspect of the recombinant host cells disclosed herein:
The invention also provides a recombinant host cell capable of producing a citronellal, a citronellol, or a citronellic acid, comprising:
wherein at least one of the genes is a recombinant gene.
The invention also provides a recombinant host cell capable of producing a citronellal, a citronellol, or a citronellic acid, comprising:
wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cells disclosed herein:
In one aspect of the recombinant host cells disclosed herein:
The invention also provides a recombinant host cell capable of producing a citronellal and/or a citronellol, comprising:
wherein the recombinant host cell further comprises a gene encoding a iridoid synthase (ISY) polypeptide or a gene encoding a enoate reductase (ENR) polypeptide; and wherein at least one of the genes is a recombinant gene.
The invention also provides a recombinant host cell capable of producing a citronellal, a citronellol, or a citronellic acid, comprising:
wherein the recombinant host cell further comprises a gene encoding a iridoid synthase (ISY) polypeptide and a gene encoding a citronellal/citronellol dehydrogenase (CiDH) polypeptide, and wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cells disclosed herein:
In one aspect of the recombinant host cells disclosed herein, the citronellal is d-citronellal, l-citronellal, or a combination thereof.
In one aspect of the recombinant host cells disclosed herein, the citronellol is d-citronellol, l-citronellol, or a combination thereof.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a bacterial cell.
In one aspect of the recombinant host cells disclosed herein, the bacterial cell is Escherichia cells, Lactobacillus cells, Lactococcus cells, Corynebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a yeast cell further comprising the deletion of one or more of ADH6, RFX1, GRE2, ARI1, GCY1, and AYR1.
In one aspect of the recombinant host cells disclosed herein, the yeast comprises:
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a yeast cell further comprising a gene encoding a heterologous NADH oxidase polypeptide.
In one aspect of the recombinant host cells disclosed herein, the heterologous NADH oxidase polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:69.
In one aspect of the recombinant host cells disclosed herein, the heterologous NADH oxidase polypeptide has the amino acid sequence set forth in SEQ ID NO:69.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a yeast cell further comprising a gene encoding a carboxylic acid reductase (CAR) polypeptide.
In one aspect of the recombinant host cells disclosed herein, the CAR polypeptide comprises a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:70.
In one aspect of the recombinant host cells disclosed herein, the CAR polypeptide has the amino acid sequence set forth in SEQ ID NO:70.
In one aspect of the recombinant host cells disclosed herein, the recombinant yeast cell further comprises a gene encoding a phosphopaneine transferase (PPTase) polypeptide.
In one aspect of the recombinant host cells disclosed herein, the PPTase polypeptide comprises a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:71.
In one aspect of the recombinant host cells disclosed herein, the PPTase polypeptide has the amino acid sequence set forth in SEQ ID NO:71
In one aspect of the recombinant host cells disclosed herein, the yeast cell is a Saccharomyces cerevisiae cell.
In one aspect of the recombinant host cells disclosed herein, the Saccharomyces cerevisiae cell contains a farnesyl pyrophosphate synthase (ERG20) gene that is transcriptionally downregulated or mutated to provide lower than wild type farnesyl pyrophosphate synthase activity.
The invention also provides a method of producing a citronellal, a citronellol, or a citronellic acid, comprising growing the recombinant host cells disclosed herein in a cell culture broth, under conditions in which the genes are expressed, wherein the citronellal, citronellol, or citronellic acid is produced by the recombinant host cell.
In one aspect of the methods disclosed herein, the recombinant host cell is transformed with one or more plasmids comprising a gene encoding the GPPS polypeptide or a gene encoding the NPPS polypeptide, a gene encoding the GES polypeptide or a gene encoding the NES polypeptide, a gene encoding the GeDH polypeptide or a gene encoding the NeDH polypeptide, a gene encoding the ISY polypeptide, a gene encoding the CiDH polypeptide, and/or a gene encoding the ENR polypeptide; wherein at least one of the genes is a recombinant gene.
In one aspect of the methods disclosed herein, the recombinant host cell is transformed with a gene encoding the GPPS polypeptide or a gene encoding the NPPS polypeptide, a gene encoding the GES polypeptide or a gene encoding the NES polypeptide, a gene encoding the GeDH polypeptide or a gene encoding the NeDH polypeptide, a gene encoding the ISY polypeptide, a gene encoding the CiDH polypeptide, and/or a gene encoding the ENR polypeptide; wherein at least one of the genes is a recombinant gene.
In one aspect of the methods disclosed herein, at least one of the recombinant genes is integrated within the host cell genome.
The invention also provides a method of producing a citronellal or a citronellol, comprising a whole-cell bioconversion of citronellal or citronellol precursors in a cell culture broth using one or more of:
wherein at least one of the polypeptides is a recombinant polypeptide; and producing the citronellal or the citronellol thereby.
In one aspect of the methods disclosed herein:
In one aspect of the methods disclosed herein:
The invention also provides a method of producing a citronellal or a citronellol, comprising:
wherein the citronellal or the citronellol is produced by the recombinant host cell.
In one aspect of the methods disclosed herein, the mevalonate pathway is endogenous or exogenous to the host cell, and is subdivided into one or more operons or coordinated gene regulation element.
In one aspect of the methods disclosed herein, a first operon of the one or more operons or coordinated gene regulation element comprises:
In one aspect of the methods disclosed herein, a second operon of the one or more operons or coordinated gene regulation element comprises:
In one aspect of the methods disclosed herein, the citronellal plasmid comprises:
In one aspect of the methods disclosed herein, the gene encoding a geranyl diphosphate synthase is Abies grandis geranyl diphosphate synthase (Ag_GPPS2) or Picea glauca geranyl diphosphate synthase (Pg_GPPS), and the gene encoding a geraniol synthase is Catharanthus roseus eraniol Synthase (Cr_GES), Ocimum basilicum geraniol synthase (Ob_GES), Phyla dulcis geraniol synthase (Pd_GES), or Valeriana officinalis geraniol synthase (VO_GES) gene.
In one aspect of the methods disclosed herein, the culture media further comprises nerol and/or geraniol.
In one aspect of the methods disclosed herein, the host cell is contacted with an oxidizing bacteria.
In one aspect of the methods disclosed herein, the oxidizing bacteria is from the genus Gluconobacter.
In one aspect of the methods disclosed herein, the oxidizing bacteria is Gluconobacter cerinus, Gluconobacter frateurii, or Gluconobacter oxydans.
In one aspect, the methods disclosed herein further comprise isolating the produced citronellal, the citronellol, or the citronellic acid alone or a combination thereof.
In one aspect of the methods disclosed herein, isolating step comprises:
In one aspect of the methods disclosed herein, isolating step comprises:
In one aspect, the methods disclosed herein further comprise recovering the citronellal, the citronellol, the citronellic acid, or a composition thereof.
In one aspect of the methods disclosed herein, the recovered composition is enriched with an optically pure composition of citronellal or citronellol.
In one aspect of the methods disclosed herein, the recombinant host cell is a bacterial cell.
In one aspect of the methods disclosed herein, the bacterial cell is Escherichia cells, Lactobacillus cells, Lactococcus cells, Cornebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
In one aspect of the methods disclosed herein, the recombinant host cell is a yeast cell.
In one aspect of the methods disclosed herein, the yeast cell is Saccharomyces cerevisiae.
The invention also provides a use of a GeDH polypeptide in the manufacture of geranial, citronellal, citronellol, citronellic acid, or a combination thereof.
The invention also provides a use of a NeDH polypeptide in the manufacture of neral, citronellal, citronellol, or citronellic acid or a combination thereof.
The invention also provides a use of a GeDH polypeptide and/or a NeDH polypeptide for the manufacture of geranial, neral, citronellal, citronellol, or citronellic acid or a combination thereof.
The invention also provides a use of a CiDH polypeptide in the manufacture of citronellal, citronellic acid or a combination thereof.
The invention also provides a use of a GeDH polypeptide in an in vitro or a whole-cell bioconversion manufacture of geranial, citronellal, citronellol, or citronellic acid or a combination thereof.
The invention also provides a use of a NeDH polypeptide in an in vitro or a whole-cell bioconversion manufacture of neral, citronellal, citronellol, or citronellic acid or a combination thereof.
The invention also provides a use of a GeDH polypeptide and a NeDH polypeptide in an in vitro or a whole-cell bioconversion manufacture of geranial, neral, citronellal, citronellol, or citronellic acid or a combination thereof.
The invention also provides a use of a CiDH polypeptide in an in vitro or a whole-cell bioconversion manufacture of citronellal, citronellic acid, or a combination thereof.
In one aspect of the uses disclosed herein, the GeDH polypeptide comprises a polypeptide having at least 45% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-6, 19, 20, or 24-32.
In one aspect of the uses disclosed herein, the NeDH polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-6, 19, 20, or 24-32.
In one aspect of the uses disclosed herein, the CiDH polypeptide comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:49-52.
The invention also provides a use of a ENR polypeptide in the manufacture of citronellol, citronellal, citronellic acid, or a combination thereof.
The invention also provides a use of a ENR polypeptide in an in vitro or a whole-cell bioconversion manufacture of citronellol, citronellal, citronellic acid, or a combination thereof.
In one aspect of the uses disclosed herein, the ENR polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:7-9, 21, 22, 33, 34, 37, 44-48, 54, 55, or 67.
The invention also provides a use of a ISY polypeptide in the manufacture of citronellol, citronellal, citronellic acid or a combination thereof.
The invention also provides a use of a ISY polypeptide in an in vitro or a whole-cell bioconversion manufacture of citronellol, citronellal, citronellic acid, or a combination thereof.
In one aspect of the uses disclosed herein, the ISY polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:54 or 55.
The invention also provides a use of a AR polypeptide in the manufacture of geraniol, nerol, citronellol, citronellic acid or a combination thereof.
The invention also provides a use of a AR polypeptide in an in vitro or a whole-cell bioconversion manufacture of geraniol, nerol, citronellol, citronellic acid or a combination thereof.
In one aspect of the uses disclosed herein, the AR polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:31, 32, or 83-86.
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:
Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
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.
As used herein, the term “about” refers to ±10% of a given value.
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. Such DNA sequences 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.
As used herein, the term “recombinant gene” or “recombinant DNA sequence” refers to a gene or DNA sequence that is not wild type in the host. Recombinant genes and recombinant DNA sequences can be introduced from another species into a recipient host, or can be derived from a wild type gene or DNA sequence such that a DNA sequence already present in the host has been augmented, modified or mutated through genetic engineering by mutagenesis and/or recombinant methods to form a recombinant host. Examples of a recombinant gene or recombinant DNA sequence include, but are not limited to, an exogenous gene introduced into a host, an endogenous gene modified or mutated so as to result in a variant displaying altered activity or functionality of the gene product, a chimeric gene (such as created by domain-swapping of proteins) a codon-optimized gene, an endogenous or heterologous gene linked to or under the control of a different transcriptional regulator such as promoter, operator, repressor or terminator. 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, such that the recombinant host comprises endogenous genes present in a higher copy number than the wild type host cell. In some embodiments, recombinant genes are synthetic and/or codon-optimized for expression in a host cell (for example, S. cerevisiae (SEQ ID NO:1-9) or E. coli (SEQ ID NO:10-22).
As used herein, the term “engineered biosynthetic pathway” refers to a biosynthetic pathway comprising at least one recombinant gene or recombinant DNA sequence 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 (wild type) 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 in a recombinant host. 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. See, e.g., Prelich, 2012, Genetics 190:841-54.
In some embodiments, an endogenous yeast gene, for example ADH, is deleted or is transcriptionally downregulated. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65. 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 term “heterologous” gene describes a gene derived from a species other than the recombinant host. In some embodiments, the recombinant host is S. cerevisiae, and a heterologous gene is derived from an organism other than S. cerevisiae. A gene 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. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). 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., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and 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 terms “mevalonate pathway”, “isoprenoid pathway” and the “HMG-CoA reductase pathway” can be used interchangeably and refer to a metabolic pathway that synthesizes isopentyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP). IPP and/or DMAPP are typically utilised by cells in the production of isoprenoids. The primary substrate for the mevalonate pathway is acetyl coenzyme A (acetyl-CoA), which is generated by cells in the breakdown of a carbon source (such as but not limited to glucose, acetate, ethanol) for example by glycolysis, or the breakdown of fatty acids through β-oxidation. IPP and DMAPP are five-carbon intermediates which when contacted with a geranyl diphosphate synthase (GPPS) together yield geranyl diphosphate (GPP). Alternatively, the IPP and DMAPP can be contacted with a neryl diphosphate synthase (NPPS) to yield neryl diphosphate (NPP).
As used herein, the term “geranyl diphosphate synthase” (GPPS), refers to an enzyme, polypeptide or fragment thereof that is able to catalyze the production of geranyl diphosphate (GPP) from IPP and DMAPP. For the avoidance of doubt, genes encoding polypeptides with farnesyl pyrophosphate synthase (FPPS) activity can also possess GPPS activity, either natively or as a result of mutation. As non-limiting examples, the FPPS genes ERG20 and of ispA can be mutated to produce enzymes with GPPS activity. For the avoidance of doubt, the term “geranyl diphosphate synthase” (GPPS) as used herein thus encompasses such mutated or otherwise recombinant ERG20 and ispA genes encoding polypeptides or enzymes possessing GPPS activity.
As used herein, the term “contact” is used to refer to any physical interaction between two objects. For example, the term “contact” can refer to the interaction between an enzyme and a substrate. In another example, the term “contact” can refer to the interaction between a liquid (e.g., a supernatant) and an adsorbent resin.
As used herein, the terms “isopentenyl pyrophosphate”, “IPP”, “isopentenyl diphosphate” and “IDP” can be used interchangeably. The term IPP refers to a product of the mevalonate pathway.
As used herein, the term “dimethylallyl pyrophosphate”, “dimethylallyl diphosphate”, “DMAPP” and “DMADP” can be used interchangeably. The term DMAPP refers to an isomer of IPP. DMAPP is isomerized from IPP by the enzyme isopentenyl pyrophosphate isomerase.
As used herein, the terms “aldehyde reductase”, “AR” and “aldose reductase” can be used interchangeably. Aldehyde reductase refers to a NAD(P)H-dependent oxidoreductase that catalyzes the reduction of aldehydes and carbonyls.
As used herein the term “citronellal/citronellol pathway” refers to the biosynthetic engineered pathway for the expression of citronellal, citronellol, and/or citronellic acid. In some aspects, the citronellal/citronellol pathway can be initiated by geraniol synthase (GES), which catalyzes the reaction of GPP or NPP to geraniol.
In some aspects, a geraniol synthase catalyzes the conversion of GPP into geraniol and in some aspects, a geraniol synthase catalyzes the conversion of GPP into geraniol. Geraniol is then oxidized by geraniol dehydrogenase (GeDH) to produce geranial. The third step of the citronellal/citronellol pathway is the reduction of geranial to citronellal via an enoate reductase (ENR) (see
Alternatively, the citronellal/citronellol pathway can be initiated by nerol synthase (NES), which catalyzes the conversion of NPP or GPP into nerol. In some aspects, a nerol synthase catalyzes the conversion of GPP into nerol and in some aspects, a nerol synthase catalyzes the conversion of GPP into nerol. Nerol is then oxidized by neral dehydrogenase (NeDH) to produce neral. Neral is then converted to l-citronellal by an enoate reductase (ENR) activity (see
As used herein, the terms “geraniol dehydrogenase” (GeDH) and “nerol dehydrogenase” (NeDH) refer to enzymes, polypeptides and fragments thereof with the ability to synthesize geranial from geraniol, and neral from nerol, respectively. In some embodiments, the same polypeptide can exhibit one or both activities. For example, the gene product of Rs_GeDH is a polypeptide exhibiting both GeDH and NeDH activities.
As used herein, the terms “citronellol precursor”, “citronellol precursors”, “citronellic acid precursors” and “citronellal/citronellol intermediates” refer to intermediates in the mevalonate pathway such IPP and DMAPP and intermediates in the citronellal/citronellol pathway, such as GPP, geraniol, geranial and/or citronellal.
In one embodiment, isomerisation of neral to geranial can happen via keto-enol tautomerization. As used herein, the term “keto-enol tautomerization” refers to the conversion of a keto form to an enol form.
The fourth step of this process reduces citronellal to citronellol by alcohol dehydrogenase/aldehyde reductase activity in the host cell. The dehydrogenase can be selected from Castellaniella defragrans geraniol dehydrogenase (Cd_GeDH), Thauera terpenica 58Eu geraniol dehydrogenase (Tt_GeDH), Spingopyxis macrogoltabida geraniol dehydrogenase (Sm_GeDH) and, Acinetobacter calcoaceticus geraniol dehydrogenase (Ac_GeDH), or other geraniol dehydrogenases not listed. The aldehyde reductase (AR) activity can be a result of endogenous or exogenous aldehyde reductase enzyme activity. Additionally, the aldehyde reductase that converts neral to nerol, geranial to geraniol, or citronellal to citronellol can be the same or different (see
Alternatively, the citronellal/citronellol pathway can produce citronellol via conversion of NPP or GPP to nerol or geraniol by NES or GES activity, respectively. In this embodiment, Enoate reductase (ENR) or Iridoid synthase (ISY) activity converts nerol or geraniol to l- or d-citronellol, respectively. D- or l-citronellol is then oxidized by citronellal/citronellol dehydrogenase (CiDH) to yield d- or l-citronellal, respectively (see
As used herein, the term “synthase” refers to an enzyme that catalyses a synthesis process typically involving the linkage of two or more molecules, (for example, the reaction in which acetyl-CoA condenses with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA is catalysed by HMG-CoA synthase.
As used herein, the term “kinase” refers to an enzyme that catalyses the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates, such as mevalonate kinase (MK) and phosphomevalonate kinase (PMK).
As used herein the terms “enantiomer”, “enantiomers”, “optical isomer”, “stereoisomer” or “optical isomers” refer to a chiral molecule or chiral molecules that are mirror images of one another. These molecules are non-superimposable on one another and are distinguished by those skilled in through the use of prefixes. There are three major nomenclature systems with equivalence as follows: the +/− optical activity system (+ and −) based on the ability of a pure isomer to rotate plane polarized light clockwise or anticlockwise; the d/l system otherwise known as the dextrorotation- and levorotation-system based on direct translation into Latin (dexter for right and laevus for left). Thus “l” is equivalent to “−”, and “d” is equivalent to “+”. The two systems are herein used interchangeably. For the avoidance of doubt, the related R/S system based on the Latin language (rectus for proper and sinister for straight) is used to characterize the absolute configuration of a specific stereocenter, of which there can be more than one in a molecule, though it can also be used to characterize an entire molecule if it comprises a single stereocenter. As used herein the term “d-enantiomer” refers to a molecule having a chiral carbon in which the higher polarity group is located on the right of the Fischer projection (D-enantiomers rotate plane polarized light clockwise (+)). As used herein the term “l-enantiomer” refers to a molecule having a chiral carbon in which the higher polarity group is located on the left of the Fischer projection (L-enantiomers rotate plane polarized light counterclockwise (−)).
As used herein, the terms “optical purity” and “enantiomeric excess” can be used interchangeably. Optical purity refers to a measure of purity used for chiral substances. For example, if the optical purity is 100% then only one enantiomer (either d- or l-) was produced. Additionally, if the pathway produces 90% d-citronellal and 10% l-citronellal then the optical purity of d-citronellal is 90%-10%=80% enantiomeric excess (ee).
As used herein, the term “isomerase” refers to an enzyme which converts a molecule from one isomer to another. An isomerase can facilitate the intramolecular rearrangement in which bonds are broken and formed or they can catalyze conformational changes, such as isopentenyl diphosphate isomerase.
As used herein, the term “reductase” refers to an enzyme that acts as a reducing agent. Reductases include but are not limited to HMG-CoA reductase and enoate reductase (ENR). As used herein, the terms “enoate reductase” and “ene reductase” are used interchangeably, and also comprise the yeast Old Yellow Enzymes (OYE) class of flavoproteins. For the avoidance of doubt, Iridoid synthases (such as, but not limited to, Oe_ISY and Cr_ISY) can possess enoate reductase activity and such enzymes are thus encompassed by the term “enoate reductase” as used herein.
As used herein, the term “derivative” refers to a molecule or compound that is derived from a similar compound by some chemical or physical process.
As used herein, the term “nerol” refers to a monoterpene with a fresh sweet rose odor originally isolated from neroli oil, but also present in essential oils from lemongrass and hops. Nerol is the cis-trans isomer of geraniol.
As used herein, the terms “citronellol” or “dihydrogeraniol” refer to a natural acyclic monoterpenoid that can be produced by the hydrogenation of geraniol (trans).
As used herein, the terms “neral” and “geranial” refer to a liquid aldehydes that are constituents of citral, an essential oil naturally derived from such plants as lemon myrtle, Litsea citrata, Litsea cubeba, lemongrass, lemon tea-tree. Citral, also known as 3,7-dimethyl-2,6-dienal or lemonal, is a blend of isomeric terpenoids in which the E-isomer (citral A) is geranial providing a strong lemon citrus odor and the Z-isomer (citral B) is neral providing sweeter but less intense lemon odor. Citral is used commercially as an antimicrobial, a fragrance, fragrance component, a flavoring agent, to fortify lemon, and in the synthesis of vitamin A.
As used herein, the term “geraniol” refers to a monoterpoenoid alcohol with a rose-like scent naturally present in rose oil, germanioum oil, palmarosa oil, lemon oil and citronella oil. Geraniol is used commercially a fragrance component in perfumes, typically with flavors such as peach, raspberry, grapefruit, red apple, plum, lime, orange, lemon, watermelon, pineapple, and blueberry.
As used herein, the terms “citronellal”, “rhodinal” or “3, 7-dimethyloct-6-en-1-al” refer to a monoterpenoid that is the main component in citronella oil and provides its distinctive lemon scent. Citronellal can be present as an l or d enantiomer.
As used herein, the term “dehydrogenase” refers to an enzyme that oxidizes a substrate by a reduction reaction that removes one or more hydrogen molecules from a substrate to an electron acceptor, such as geraniol dehydrogenase or alcohol dehydrogenase.
As used herein, the terms “mevalonate plasmid” and “pMev” can be used interchangeably. The term mevalonate plasmid refers to a plasmid transformed into E. coli that can result in the production of IPP and DMAPP from acetyl-CoA and/or malonyl-CoA. The genes included in the mevalonate plasmid can be endogenous or exogenous. The first three genes; Escherichia coli MG1655 Acetyl-CoA acetyltransferase (Ec_atoB), Staphylococcus aureus HMG-CoA synthase (Sa_mvaS), Staphylococcus aureus HMG-CoA reductase (Sa_mvaA) are under control of one promoter and the last four genes: Saccharomyces cerevisiae Mevalonate Kinase (Sc_erg12), Saccharomyces cerevisiae Phosphomevalonate kinase (Sc_erg8), Saccharomyces cerevisiae Diphospomevalonate decarboxylase (Sc_erg19), and Escherichia coli Isopentenyl diphosphate isomerase (Ec_idi) are under control of another promoter. Each operon is terminated by a transcriptional terminator. The seven genes are located on a p15-based replicative plasmid backbone and the kanamycin selection marker. All genes except Ec_IDI are heterologous and were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as Escherichia coli codon optimized variants. Ec_atoB was a recombinant gene produced by codon optimization for E. coli of the endogenous gene of the E. coli wild type host (the wild type gene was modified because it did not extensively use the preferred codons for expression in E. coli).
As used herein, the terms “citronellal plasmid”, “pCitro plasmid” and “pCitro” can be used interchangeably. The term citronellal plasmid refers to a plasmid that following transformation into a host cells can result in the production of citronellal from IPP and DMAPP. The genes in the citronellal plasmid can be either endogenous or exogenous. In one non-limiting example disclosed herein, the 4 enzymes: geranyl diphosphate synthase, geraniol synthase, ene reductase and geraniol dehydrogenase encoded by the heterologous genes Ag_GPPS2, Cr_GES, KI_KYE1 (alternatively Ps_OYE2.6) and Cd_GeDH (alternatively Rs_GeDH), were subdivided on two operons. The first three genes are under the control of a promoter and terminated by a transcriptional terminator. The last gene is under the control of another promoter and terminator. The 4 genes are located on a pBR322-based replicative plasmid backbone and ampicillin selection marker, though any suitable plasmid (preferably a high copy number plasmid) and selection marker can be used. All genes were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany). Since the species of host cell in this instance was chosen to be Escherichia coli, the genes in this example were codon optimized variants (except Rs_GeDH which was codon optimized for Saccharomyces cerevisiae).
As used herein, the terms “operon” and “operons” and “coordinated gene regulation element” are functionally equivalent, are used interchangeably, and refer to a genetic regulator system comprising a functioning unit of DNA containing more than one gene under the control of a single promoter. Although highly prevalent as a means of coordinated gene regulation, operons are not ubiquitous. For example, in yeast (such as Saccharomyces cerevisiae) coordinated gene regulation is frequently achieved by linking opposite DNA strand genes using a common promoter element, or by concatenating more than one functional domain into a single peptide to provide chimeric proteins. All of the above systems using a single promoter to regulate more than one functional activity are observed in wild type host cells of one species or another, and have inspired the engineering of multi-enzyme pathways under coordinated expression using recombinant DNA techniques.
As used herein, the term “plasmid” or “plasmids” refer to a small, circular, double-stranded DNA molecule that is distinct from the chromosomal DNA of a cell. Plasmids have a wide range of lengths and offer several genetic advantages. Plasmids are one form of “vector” and are particularly useful for introducing and maintain foreign (exogenous) DNA within a host cell. Choice of plasmid for a particular application is typically dictated by the ability to be maintained in one or more host cell species at a chosen copy number of plasmids per host cell.
As used herein, the terms “acetyltransferase” and “transacetylase” can be used interchangeably. “Acetyltransferase” (such as acetyl-CoA acetyltransferase) refers to a type of transferase that transfers an acetyl group from an acetyl-CoA to a recipient compound, such as for example, a lysine amino acid.
Exemplary UniProt Numbers for specific embodiments of such enzymes include: H1ZV38 (SEQ ID NO:1), AOAOE4B3N6 (SEQ ID NO:2), A0A0P0DQG4 (SEQ ID NO:3), Q59096 (SEQ ID NO:4), C9E0G2 (SEQ ID NO:5), D5MPF3 (SEQ ID NO:6), P40952 (SEQ ID NO:7), A3LT82 (SEQ ID NO:8), Q9FEW9 (SEQ ID NO:67), Q03558 (SEQ ID NO:33), G1FCG0 (SEQ ID NO:34), Q88NF7 (SEQ ID NO:37), Q5NLA1 (SEQ ID NO:9), Q6I7B7 (SEQ ID NO:44), G6XL43 (SEQ ID NO:45), A0A0D6MPY3 (SEQ ID NO:46), F8EUA7 (SEQ ID NO:47), B7L5K3 (SEQ ID NO:48, Q1WF68 (SEQ ID NO:49), Q1WF63 (SEQ ID NO:50), J1IP19 (SEQ ID NO:51), U1H7S9 (SEQ ID NO:52), C1K5M2 (SEQ ID NO:53), A0A0U3J294 (SEQ ID NO:54), K7WDL7 (SEQ ID NO:55), R4HEK6 (SEQ ID NO:56), T2DP90 (SEQ ID NO:57), J7JYU1 (SEQ ID NO:58), P76461 (SEQ ID NO:10), Q9FD87 (SEQ ID NO:11), Q9FD86 (SEQ ID NO:12), P07277 (SEQ ID NO:13), P24521 (SEQ ID NO:14), P32377 (SEQ ID NO:15), Q46822 (SEQ ID NO:16), P40952 (SEQ ID NO:7), A3LT82 (SEQ ID NO:22), A0A0M2H8A0 (SEQ ID NO:30), P27250 (SEQ ID NO:31), P75691 (SEQ ID NO:32), B2N194 (SEQ ID NO:24), D2WKD9 (SEQ ID NO:25), Q2KNL5 (SEQ ID NO:26), AOAOE4B3N6 (SEQ ID NO:27), C9E0G2 (SEQ ID NO:28), A0A0X8R1M5 (SEQ ID NO:29), J9PZR5 (SEQ ID NO:18), Q6USK1 (SEQ ID NO:63), E9JGT2 (SEQ ID NO:64), C0KWV4 (SEQ ID NO:65), V9ZAD7 (SEQ ID NO:66), Q04894 (SEQ ID NO:68).
In one embodiment, the present invention contemplates in vivo and in vitro production of one or more of citronellal, citronellol, citronellic acid, or citronellal precursors. In a further embodiment, the present invention contemplates a combination of in vivo and in vitro steps for the production one or more of citronellal, citronellol, citronellic acid, or citronellal precursors. In one particular embodiment, the present invention provides recombinant hosts containing an engineered biosynthetic pathway capable of producing one or more of citronellal, citronellol, citronellic acid, or citronellal precursors, said engineered biosynthetic pathway including one or more expressed and functional heterologous enzymes.
For example, in some aspects the present invention provides recombinant microorganism (such as a yeast or bacterial) cells capable of producing in vivo citronellol precursors. In particular, recombinant yeast or bacterial cells as provided herein are capable of expressing one or more dehydrogenases and/or other proteins capable of converting geraniol to geranial and citronellal to citronellol. Sources for dehydrogenases include but are not limited to bacteria, including several species of Rhizobium, Streptomyces, Pseudomonas, Escherichia and Bacillus that naturally express these enzymes. In other particular embodiments, dehydrogenases used herein can be derived from yeast, fungi, plants, and/or animals.
In another embodiment, the invention provides recombinant microorganism (such as a yeast or bacterial) cells capable of expressing one or more reductases and/or other proteins capable of converting geranial to citronellal.
In another embodiment, the invention provides recombinant microorganism (such as a yeast or bacterial) cells capable of expressing one or more reductases and/or other proteins capable of converting neral to citronellal.
In another embodiment, the invention provides recombinant microorganism (such as a yeast or bacterial) cells capable of expressing one or more synthases and/or other proteins capable of converting IPP and DMAPP to GPP or GPP to geraniol.
In another embodiment, the invention provides recombinant microorganism (such as a yeast or bacterial) cells capable of expressing one or more synthases and/or other proteins capable of converting IPP and DMAPP to NPP or NPP to nerol.
In another embodiment, the recombinant microorganism (such as a yeast or bacterial) cells capable of producing citronellol precursors can be further modified to increase citronellol precursor production by increasing IPP and DMAPP levels via the replacement of the native farnesyl pyrophosphate synthase promoter with a weaker promoter, resulting in the transcriptional downregulation of the native farnesyl pyrophosphate synthase. As a non-limiting example, the yeast ERG20 gene encodes the yeast farnesyl pyrophosphate synthase, which acts to catalyse the formation of farnesyl diphosphate from GPP and IPP, and it is possible to increase IPP and DMAPP in yeast by replacing the ERG20 promoter with the KEX2 promoter, resulting in the transcriptional downregulation of ERG20.
In another embodiment, the recombinant microorganism (such as a yeast or bacterial) cells with elevated levels of IPP and DMAPP are well suited for the introduction and/or integration of pathway expression cassettes for the genes necessary to yield citronellal and/or citronellol. For example, Ag_GPPS2 can be under the control of the TEF1 promoter, Cr_GES can be under the control of PGK1 promoter, Rs_GeDH can be under the control of PGK1 promoter and KI_KYE1 can be under the control of the TPI1 promoter. In some aspects, expression cassettes can contain flanking regions homologous to regions of the host genome, so as to allow targeted integrated in the host cell genome (for example Saccharomyces cerevisiae or Escherichia coli) by homologous recombination (see e.g., WO 2014/027118 which is incorporated by reference in its entirety).
In another embodiment, elevated levels of IPP and DMAPP can be achieved in E. coli host cells using one or more operons collectively comprising genes required for the mevalonate pathway having one or more mevalonate pathway genes optimized for E. coli. In a certain embodiment, the seven genes comprising the mevalonate pathway can be present on one or more plasmids and/or integrated into the genomic DNA of the host. For example, in one embodiment the mevalonate pathway can be introduced and maintained in E. coli subdivided into two operons on a single plasmid: Ec_atoB, Sa_mvaS, and Sa_mvaA can be driven by one promoter and Sc_erg12, Sc_erg8, Sc_erg19 and Ec_idi can be driven by another promoter. In this instance, all mevalonate pathway genes except Ec_atoB are heterologous genes optimized for E. coli. Optimization of genes for heterologous expression in a particular host species typically makes use of the understanding of preferred codon usage patterns.
In another embodiment, recombinant protein expression of citronellal, citronellol and/or citronellic acid in E. coli can occur via a citronellal plasmid comprising E. coli optimized genes. For example, transcription of Ag_GPPS2, Cr_GES, KI-KYE1 and Cd_GeDH can be driven by one or more promoters. In some embodiments, the promoters are constitutive promoters. A constitutive promoter refers to a promoter not regulated by transcription factors, that allows for continual transcription of the coding sequence or gene under its control. Examples of constitutive promoters include, but are not limited to, PT7, PTrc, PTac and PLac without their operator, PGapA, PGadE). In other embodiments, the promoters are inducible promoters that allow for chemical or physical transcriptional regulation of the gene under regulation. A positively regulated inducible promoter refers to a promoter that allows for elevated transcription of the coding sequence or gene under its control in the presence of a biotic or abiotic factor. In further embodiments, induction of the gene to higher rates of transcription can be induced by the addition of a factor that inactivates a transcriptional repressor molecule. In yet further embodiments, activators and repressors can function in multi-regulated inducible promoters. Examples of inducible promoters include, but are not limited to, alcA.
In another embodiment, recombinant protein expression of citronellal, citronellol and/or citronellic acid in E. coli can occur via a citronellal plasmid comprising one or more E. coli optimized genes. Transcription of Ag_tGPPs, Cr_GES, Ps_OYE2.6 and Rs_GeDH can be driven by one or more promoters, such as the trc-promoter. In one example, the Ag_tGPPs, Cr_GES and Ps_OYE2.6 genes are optimised for E. coli.
For example, the present invention provides recombinant microorganism (such as a yeast or bacterial) cells capable of producing in vivo one or more of citronellal, citronellol or citronellic acid. In particular, recombinant yeast or bacterial cells as provided herein are capable of expressing one or more dehydrogenases, reductases and/or other proteins capable of converting geraniol to citronellal, citronellol and/or citronellic acid.
In another embodiment, the recombinant microorganism (such as a yeast or bacterial) cells disclosed herein are capable of expressing one or more geraniol dehydrogenases capable of catalysing the formation of geraniol to geranial, and/or citronellal to citronellol.
In a further embodiment, the recombinant microorganism (such as a yeast or bacterial) cells disclosed herein are capable of expressing one or more ene reductases capable of reducing geranial to citronellal.
As used herein, the terms “detectable amount,” “detectable concentration,” “measurable amount,” and “measurable concentration” refer to a level of a specific product to be measured (for example, geraniol, geranial, citronellal, citronellol, citronellic acid, or citronellal/citronellol intermediates and/or citronellal/citronellol precursors). The product can be measured in AUC, μM/OD600, mg/L, μM, or mM. Geraniol, geranial, citronellal, citronellol, citronellic acid, citronellal/citronellol intermediate and/or citronellal/citronellol precursor production (i.e., total, supernatant, organic phase, and/or intracellular geraniol, geranial, and/or citronellal levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/spectrophotometry (UV-Vis), mass spectrometry (MS), and NMR.
As used herein, the term “undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound at an “undetectable concentration” (<1 ppm) is not present.
As used herein, the terms “or” and “and/or” is utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” In some embodiments, “and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In certain embodiments, “and/or” is used to refer to production of geraniol, geranial, citronellal and/or citronellol, wherein one or more geraniol, geranial, citronellal and/or citronellol are produced. In yet another embodiment, “and/or” is used to refer to production of geraniol, geranial, citronellal, citronellol and/or citronellic acid wherein one or more geraniol, geranial, citronellal, citronellol and/or citronellic acid are produced through one or more of the following steps: culturing a recombinant microorganism, producing one or more geraniol, geranial, citronellal, citronellol and/or citronellic acid in a recombinant microorganism, and/or isolating one or more geraniol, geranial, citronellal citronellol and/or citronellic acid.
Functional Homologs
Functional homologs of the polypeptides described herein are also suitable for use in producing citronellol, citronellal, citronellic acid and/or precursors thereof in a recombinant host.
A functional homolog refers to 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 citronellol and citronellol precursor biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using any sequence disclosed herein as a reference sequence for a database search for homologs. Amino acid sequence can be, 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 citronellol and citronellol precursor 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 citronellol and citronellol precursor 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 citronellol and citronellol precursor biosynthesis polypeptides 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/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). 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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity.
For example, polypeptides suitable for producing citronellol and/or citronellol precursors in a recombinant host include functional homologs of Ag_GPPS2, Cr_GES, KI_KYE1, Cd_GeDH, Tt_GeDH, Rs_GeDH, Sm_GeDH, Ac_GeDH, Pp_GeDH, Ps_OYE2.6, Zm_OYE, Ec_atoB, Sa_mvaS, Sa_mvaA, Sc_erg12, Sc_erg8, Sc_erg19, Lc_MVA, Ef_MVA, Sp_IDI, ScIDI and Ec_idi.
Methods to modify the substrate specificity of, 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 Labrou N E., Curr Protein Pept Sci. 11(1):91-100.
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 an amino acid sequence described herein) is aligned to one or more candidate sequences using a computer program (for example, ClustalW (version 1.83, default parameters), or the Needleman-Wunsch algorithm), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res. 31(13):3497-500.
Clustal Omega 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: percentage; 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: percentage; 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, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The Clustal Omega output is a sequence alignment that reflects the relationship between sequences. Clustal Omega 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_www.ebi.ac.uk/Tools/msa/clustalo/.
To determine a percent identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, 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.
To determine a percent identity of a candidate 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.
It will be appreciated that geraniol dehydrogenase, geraniol dehydrogenase-like proteins, enoate (ene) reductase, ene reductase-like proteins, acetyl-CoA acetyltransferase and acetyl-CoA acetyltransferase-like proteins, HMG-CoA synthase and HMG-CoA synthase-like proteins, mevalonate kinase and mevalonate kinase-like proteins, phosphomevalonate kinase and phosphor mevalonate kinase-like proteins, isopentenyl diphosphate isomerase and isopentenyl diphosphate isomerase-like proteins, geranyl diphosphate synthase and geranyl diphosphate synthase-like proteins, geraniol synthase and geraniol synthase-like proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes.
It will be appreciated that functional dehydrogenase, reductase, synthase, acetyltransferase, kinase, decarboxylase and isomerase proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. 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 proteins engineered through the joining of two or more genes that code for different proteins.
In some embodiments, a nucleic acid sequence encoding a geraniol dehydrogenase, an ene reductase, a geranial diphosphate synthase, a HMG-CoA synthase, a HMG-CoA reductase, an acetyl-CoA acetyltransferase, a phosphomevalonate kinase, a mevalonate kinase, di phosphomevalonate decarboxylase or an isopentenyl diphosphate isomerase polypeptide can include a tag sequence that encodes a “tag” designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), solubility, 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), disulfide oxiodoreductase (DsbA), maltose binding protein (MBP), N-utilization substance (NusA), small ubiquitin-like modifier (SUMO), 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.
Citronellol and Citronellol Precursor Biosynthesis Nucleic Acids
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 typically are integrated at positions other than the position where the native sequence is found. “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 can 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 can 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 citronellal/citronellol 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 citronellal/citronellol biosynthesis gene cluster, or a mevalonate pathway 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, citronellal/citronellol gene cluster can be combined such that each citronellal/citronellol coding sequence is operably linked to a separate regulatory region, to form a citronellal/citronellol module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for citronellal/citronellol 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 citronellol or citronellol precursor biosynthesis. For example, it can be desirable to downregulate synthesis of farnesyl pyrophosphate in a yeast strain in order to further increase IPP and DMAPP production necessary to produce GPP, e.g., by downregulating farnesyl pyrophosphate synthase. In such cases, a nucleic acid that overexpresses the polypeptide or gene product can 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.
In one embodiment, the GPPS polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:17, or 59-62 or has an amino acid sequence which has at least 50% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the GES polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:18, or 63-66 or has an amino acid sequence which has at least 40% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the GeDH polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence of any one of SEQ ID NO:1-6, 19, 20, or 24-32 or has an amino acid sequence which has at least 45% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the ENR polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:7-9, 21, 22, 33, 34, 37, 44-48, or 67 or has an amino acid sequence which has at least 50% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the AR polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:31, 32, or 83-86 or has an amino acid sequence which has at least 85% identity therewith, preferably 90% identity therewith, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the NPPS polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:53, 74, or 75, or has an amino acid sequence which has at least 45% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment the NES polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:56-58 or 77-79 has an amino acid sequence which has at least 45% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment the NeDH polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NOs:1-6, 19, 20, or 24-32 has an amino acid sequence which has at least 50% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment the ADH polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in SEQ ID NO:68, or has an amino acid sequence which has at least 45% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the ISY polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:54 or 55, or has an amino acid sequence which has at least 50% identity therewith, preferably at least 75% identity therewith, preferably at least 80%, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
In one embodiment, the CiDH polypeptide for use in any one of the recombinant host cells, methods and/or uses of the present invention has an amino acid sequence shown in any one of SEQ ID NO:49-52, or has an amino acid sequence which has at least 80% identity therewith, preferably at least 85%, preferably at least 95%, preferably at least 98% identity therewith.
Host Microorganisms
Recombinant hosts can be used to express polypeptides for producing citronellal, citronellol, citronellic acid and/or their precursors, including fungal, bacterial, yeast, mammalian, insect, and plant.
A number of prokaryotes and eukaryotes are particularly suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria (such as E. coli), yeast (such as S. cerevisiae), and fungi. A species and strain selected for use as a citronellal and citronellol precursor 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).
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 production of citronellal, citronellol, citronellic acid, and/or their precursors. 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 can also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, geranyl diphosphate, geranial, geraniol, citronellal, citronellol, and/or citronellic acid can be determined by extracting samples from culture media for analysis according to published methods.
Carbon sources of use in the instant method can include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the citronellal/citronellol. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, acetate, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbon sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose can be preferred. The carbon source can be provided to the host organism according to any feeding regimen commonly used by those skilled in the art of culturing the appropriate host cell species.
After the recombinant microorganism has been grown in culture for a period of time, wherein the culturing conditions and period of time facilitate production of citronellal, citronellol, citronellic acid, and/or one or more citronellal/citronellol precursors which can then be recovered from the culture medium, off-gas and/or recombinant microorganisms using various techniques known in the art. For example, citronellal, citronellol, citronellic acid, and their precursors diffuse and/or are transported out of the host cell by host transporters. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, citronellal, citronellol, citronelic acid, and their precursors can be trapped outside by a solvent phase (of which isopropylmyristate, IPM, is a non-limiting example) added directly to the culture medium, or they can be trapped in solvent phase (of which IPM is again a non-limiting example) in a collection container in an off-gas trapping system connected to the fermenter.
A non-limiting example of an off-gas trapping system comprises leading the off gas from the headspace above the fermentation medium in the fermenter into a cooling unit (such as a Dimroth condenser) that cools the temperature of the off-gas to below the condensation point of water vapor and citronellal or citronellol gas. The condensed water, citronellol and/or citronellal is drained and/or pumped into a collection container containing a solvent phase (such as IPM) that traps citronellal and/or citronellol. The condensed water vapor is present in the collection container as a second phase (aqueous phase), which can be drained periodically or continuously so as to increase the citronellol and/or citronellol content in the collection container. In some embodiments, the gaseous headspace of the collection container is attached to a second cooling unit (such as a Dimroth condenser) that cools any citronellal and/or citronellol gas and water vapor that escaped condensation in the first cooling unit, and collects the condensates in a second collection container containing a solvent phase and an aqueous phase. Citronellal and/or citronellol can then be enriched, purified or isolated from the solvent phase by evaporation of the solvent or precipitation (such as by temperature or pH adjustment).
In another embodiment, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant, which can then be applied to a chromatography column (e.g., a C-18 column), washed with water to remove hydrophilic compounds, then elution of the compound(s) of interest performed with a suitable solvent (a non-limiting example of which is methanol). The compound(s) can then be further purified by preparative HPLC, (relevant techniques of which are taught in, 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 or host cells rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate citronellal, citronellol, and/or citronellic acid.
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., IPP and DMAPP, can be introduced into second culture medium to be converted into a subsequent intermediate such as GPP, or into an end product such as, for example, one or more of citronellal, citronellol or citronellic acid. For example, citronellol produced by recombinant microorganisms (such as the recombinant yeast or E. coli taught herein) can be contacted with oxidizing bacteria (for example, the Gluconobacter sp., e.g., Gluconobacter oxydans, Gluconobacter cerinus, or Gluconobacter frateurii) to permit the bioconversion of citronellol into citronellal and/or citronellic acid. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host can be grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
As used herein, the terms “bioconversion” or “biotransformation” refer to the conversion of organic materials, into usable products by biological processes or agents, such as certain microorganisms. In some embodiments, citronellal, citronellol, or citronellic acid, can be produced by bioconversion using oxidizing bacteria. For bioconversion to occur, an oxidizing bacteria modifies a precursor, and/or an intermediate thereof, to the citronellol, citronellal, or citronellic acid produced by a recombinant host cell expressing one or more enzymes involved in the citronellal/citronellol pathway. Following modification in vivo, the citronellol, citronellal, or citronellic acid remains in the cell and/or is excreted into the culture medium. For example, a recombinant host cell comprising an operative engineered biosynthetic pathway, comprising: a gene encoding a geranyl diphosphate synthase (GPPS) polypeptide; a gene encoding a geraniol synthase (GES) polypeptide; a gene encoding a geraniol dehydrogenase (GeDH) polypeptide; a gene encoding a enoate reductase (ENR) polypeptide; and a gene encoding aldehyde reductase (AR) polypeptide wherein the recombinant host cell is capable of producing one or more of citronellol, citronellal, or citronellic acid is contacted with oxidizing bacteria to convert citronellol to citronellal and/or citronellic acid. In certain embodiments, bioconversion is regarded as an advantage because an efficient self-sustained bioconversion system can significantly lower the scale-up costs.
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 Abies, Acinetobactor, Castellaniella, Catharanthus, Gluconobacter, Escherichia, Kluyveromyces, Pichia, Pseudomonas, Rhodococcus, Saccharomyces, Staphylococcus, Sphingopyxis, Thauera, or Zymomonas. Exemplary species from such genera include Abies grandis, Acinetobacter calcoaceticus, Castellaniella defragrans, Catharanthus roseus, Gluconobacter oxydans, Gluconobacter cerinus, Gluconobacter frateurii, Escherichia coli, Kluyveromyces lactis, Pichia stipitis, Pseudomonas putida, Rhodococcus sp. RD6.2, Saccharomyces cerevisiae, Staphylococcus aureus, Sphingopyxis macrogoltabida, Thauera terpenica 58Eu, and Zymomonas mobilis subsp. mobilis.
In some embodiments, a microorganism can be a prokaryote, such as bacteria, for example, Escherichia coli, Lactobacillus, Lactococcus, Cornebacterium, Acetobacter, Acinetobacter, or Pseudomonas.
In other embodiments, a microorganism can be a fungus, such as an Ascomycete, for example, Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, or Saccharomyces cerevisiae.
In certain embodiments, a microorganism can be an algal cell, for example, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, or Scenedesmus almeriensis species.
In some embodiments, a microorganism can be a cyanobacterial cell, for example, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, or Scenedesmus almeriensis.
Saccharomyces spp.
Saccharomyces is a well-studied and widely used chassis 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.
Abies grandis
Abies grandis is a fir native to the Pacific Northwest and Northern California of North America, occurring at altitudes of sea level to 1,800 m. It is a major constituent of the Grand Fir/Douglas Fir Ecoregion of the Cascade Range. The tree typically grows to 40-70 m in height. There are two varieties, the taller coast grand fir, found west of the Cascade Mountains, and the shorter interior grand fir, found east of the Cascades.
Aspergillus spp.
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 citronellol and citronellol precursors.
E. coli
E. coli, another widely used platform 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.
Castellaniella defragrans
Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification.
Acinetobacter calcoaceticus
Acinetobacter calcoaceticus is a non-motile, gram negative coccobacillus. It grows under aerobic conditions, is catalase positive and oxidase negative.
Agaricus, Gibberella, and Phanerochaete spp.
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 citronellol are already produced by endogenous genes. Thus, modules comprising recombinant genes for citronellol 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 sp.
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 spp.
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.
Catharanthus roseus
Catharanthus roseus is a species of flowering plant in the dogbane family Apocynaceae. It is native and endemic to Madagascar, but grown elsewhere as an ornamental and medicinal plant, a source of the drugs vincristine and vinblastine, used to treat cancer.
Rhodococcus sp.
Rhodococcus is a genus of aerobic, nonsporulating, nonmotile Gram-positive bacteria closely related to Mycobacterium and Corynebacterium. Though a number of species have been shown to have pathogenic properties, many are benign, and have been found to survive in a wide range of environments, including soil, water, and eukaryotic cells.
Staphylococcus aureus
Staphylococcus aureus is a gram-positive coccal bacterium that is a member of the Firmicutes, and is frequently found in the nose, respiratory tract, and on the skin. It is often positive for catalase and nitrate reduction. Although S. aureus is not always pathogenic, it is a common cause of skin infections such as abscesses, respiratory infections such as sinusitis, and food poisoning.
Zymomonas mobilis
Zymomonas mobilis is a Gram negative, facultative anaerobic, non-sporulating, polarly-flagellated, rod-shaped bacterium. It has notable bioethanol-producing capabilities, which surpass yeast in some aspects. It was originally isolated from alcoholic beverages like the African palm wine, the Mexican pulque, and also as a contaminant of cider and beer (cider sickness and beer spoilage) in European countries.
Pseudomonas putida
Pseudomonas putida is a Gram-negative, rod-shaped, saprotrophic soil bacterium. It demonstrates a very diverse metabolism, including the ability to degrade organic solvents such as toluene. This ability has been put to use in bioremediation, or the use of microorganisms to biodegrade oil.
Gluconobacter sp.
Gluconobacter sp are gram-negative rod or oval shaped bacteria. They tend to have a small genome size and limited metabolic abilities. These abilities include partially oxidizing carbohydrates and alcohols through the process of oxidative fermentation. They are obligately aerobic, and have a strict respiratory type of metabolism with oxygen as the terminal electron acceptor. Gluconobacter strains prefer sugar-enriched environments. Examples include, but are not limited to, Gluconobacter oxydans, Gluconobacter cerinus, and Gluconobacter frateurii.
Citronellal/Citronellol Compositions
Significant agricultural resources in terms of land, equipment, and biomass generation are required to meet current industry needs for citronellol, citronellal, and citronellic acid. It is therefore desirable to have the ability to obtain scalable amounts of highly pure citronellol, citronellal, and citronellic acid. Recombinant hosts described herein can produce compositions that are selectively enriched for citronellol, citronellal, and/or citronellic acid. As used herein, the term “enriched” is used to describe a citronellol, citronellal, and/or citronellic acid composition with an increased proportion of a particular citronellol, citronellal, and/or citronellic acid, compared to citronellol, citronellal, and/or citronellic acid (extract) from the oils of plants such as Corymbia citriodora, Cymbopogon nardus, and Cymbopogon winterianus. Thus, the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the profile desired for a given product and that have a proportion of each citronellol, citronellal, and/or citronellic acid that is consistent from batch to batch. In some embodiments, recombinant hosts described herein do not produce or produce a reduced amount of an undesired citronellol, citronellal, and/or citronellic acid precursor and/or intermediate or by-product found in plant extracts. Thus, compositions comprising citronellol, citronellal, and/or citronellic acid produced by the recombinant hosts described herein are distinguishable from compositions derived from plants. In some embodiments, a citronellol, citronellal, and/or citronellic acid composition can be produced in vitro, in vivo, or by bioconversion.
The amount of an individual desired product (e.g., citronellol, citronellal, or citronellic acid) accumulated can be from about 1 to about 7,000 mg/L, e.g., about 1 to about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1,500 mg/L, or about 200 to about 1,000 mg/L, at least about 1,000 mg/L, at least about 1,200 mg/L, at least about at least 1,400 mg/L, at least about 1,600 mg/L, at least about 1,800 mg/L, at least about 2,800 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of citronellol, citronellal, or citronellic acid can exceed 7,000 mg/L. The amount of a combination of citronellol, citronellal, and citronellic acid accumulated can be from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1,500, at least about 2,000 mg/L, at least about 3,000 mg/L, at least about 4,000 mg/L, at least about 5,000 mg/L, at least about 6,000 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of a combination of citronellol, citronellal, and citronellic acid can exceed 7,000 mg/L. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant host microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.
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 citronellol, citronellal, and/or citronellic acid. For example, a first microorganism can comprise one or more biosynthesis genes for producing a citronellol, citronellal, and/or citronellic acid precursor, while a second microorganism comprises citronellol, citronellal, and/or citronellic acid 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., IPP and/or DMAPP, or geraniol or nerol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as citronellal. 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.
Citronellol, citronellal, and/or citronellic acid and compositions obtained by the methods disclosed herein can be used to make any number of commonly used products in the fragrance industry, such as an insect repellent, and they can also be used as an intermediate in the synthesis of several natural terpenoids. For example, substantially pure citronellol, citronellal, and/or citronellic acid can be included in products such as candles, lotions, perfumes, deodorants, toothpaste, chewing gum and oils. Substantially pure citronellol, citronellal, and/or citronellic acid can also be included. Alternatively, a mixture of citronellol, citronellal, and/or citronellic acid can be made by culturing recombinant microorganisms separately, each producing a specific citronellol, citronellal, and/or citronellic acid, recovering the citronellol, citronellal, and/or citronellic acid in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion. The recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current citronellol, citronellal, and/or citronellic acid products.
Compositions produced by a recombinant microorganism described herein can be incorporated into a number of products. For example, a citronellol, citronellal, and/or citronellic acid compositions produced by a recombinant microorganism can be incorporated into a product in an amount ranging from about 20 mg citronellol, citronellal, and/or citronellic acid/kg of product to about 1800 mg citronellol, citronellal, and/or citronellic acid/kg of product on a dry weight basis, depending on the type of citronellol, citronellal, and/or citronellic acid and product. For example, a citronellol composition can have from 90-99 weight % citronellol and an undetectable amount of plant-derived contaminants, and be incorporated into a product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
A citronellal composition can be a citronellal composition having greater than 3 weight % citronellal and be incorporated into the product such that the amount of citronellal in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the citronellal composition has an undetectable amount of plant-derived contaminants.
A citronellic acid composition can be a citronellic acid composition having greater than 3 weight % citronellic acid and be incorporated into the product such that the amount of citronellic acid in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the citronellic acid composition has an undetectable amount of plant-derived contaminants.
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.
Isopropyl myristate (IPM) was recovered from biphasic culture by centrifugation at 4000 g for 5 minutes. 20 μl of IPM was diluted 20× in hexane before quantification. 2 μl of diluted sample was injected on a Waters Acquity UPC2 system (Milford, USA) coupled to a Waters Acquity UPC2 PDA eLambda detector. Separation of the compounds was achieved on a Waters Acquity UPC2 HSS C18 SB column (1.8 μm, 3.0 mm×100 mm), kept at 40° C. Mobile phases A and B were CO2 and acetonitrile, respectively. A flow of 2.0 ml/min was used. The gradient profile was as follow: 0.2 min constant at 1% B, a linear gradient from 1% B to 10% B in 1.8 min, a wash for 1 min at 10% B and back to the initial condition. The Automatic Back-Pressure Regulator (ABPR) pressure was kept at 2000 psi on the Water Acquity CO2 convergence manager.
Monoterpenes, including citronellal, geranial, citronellol and geraniol, were analyzed by recording their UV 210 nm absorbance. Detected monoterpene compounds were quantified using a linear calibration curve with authentic standards (ranging from 0.625 mg/l to 320 mg/l; Sigma-Aldrich, Buchs, Switzerland) using Waters TargetLynx software.
100 μl in vitro assay samples were extracted by liquid-liquid extraction with 300 μl Methyl tert-butyl ether (MTBE) or hexane. MTBE or hexane was recovered by quick centrifugation at 12000×g and placed in injection vials prior analysis.
1 μl sample was injected on an Agilent 7890A GC system (Santa Clara, USA) equipped with an Agilent flame ionization detector. Separation of the compounds was achieved on a Restek Rtx®-Wax column (30 m×0.25 mm, 0.25 μm film thickness). The oven temperature was initially held at 50° C. for 0.6 min, raised to 180° C. at 20° C./min, them programmed from 180° C. to 250° C. at 60° C./min and finally held at 250° C. for 0.6 min. Hydrogen was used as carrier gas with a constant flow of 2 ml/min. The injector and detector were held at 250° C. and 260° C., respectively.
Monoterpenes, including citronellal, geranial, citronellol and geraniol, were quantified using a linear calibration curve with authentic standards (ranging from 0.078 mg/l to 40 mg/l; Sigma-Aldrich, Buchs, Switzerland) using Agilent Masshunter Quantitative Analysis software.
Recombinant yeast strains capable of producing citronellal/citronellol pathway intermediates and citronellol were engineered using precursor strains (see e.g., WO 2014027118, which is incorporated by reference in its entirety) by incorporating one or more copies of a recombinant gene encoding an Abies grandis GPPS polypeptide (SEQ ID NO:17), a recombinant gene encoding a Catharanthus roseus GES polypeptide (SEQ ID NO:18), a recombinant gene encoding a Kluyveromyces lactis ENR polypeptide (SEQ ID NO:21), Pichia stipitis ENR polypeptide (SEQ ID NO:8), Zymomonas mobilis ENR polypeptide (SEQ ID NO:9), a recombinant gene encoding a recombinant Castellaniella defragrans GeDH polypeptide (SEQ ID NO:1, SEQ ID NO:19), a recombinant gene encoding a Thaurera terpenica 58Eu GeDH polypeptide (SEQ ID NO:5), a recombinant gene encoding a Rhodococcus sp. RD6.2 GeDH polypeptide (SEQ ID NO:2), a recombinant gene encoding a Sphingopyxis macrogoltabida GeDH polypeptide (SEQ ID NO:3), a recombinant gene encoding an Acinetobacter calcoaceticus GeDH polypeptide (SEQ ID NO:4), and/or a recombinant gene encoding a Pseudomonas putida GeDH polypeptide (SEQ ID NO:6).
An exemplary heterologous or endogenous pathway in yeast and bacteria for the production of d- or l-citronellal and d- or l-citronellol is shown in
An additional aspect of the citronellal/citronellol pathway can be the isomerization of geranial to neral. This step can occur chemically due to keto-enol tautomerization. Neral can then be converted to nerol by AR activity in the host cell in addition to reverse activity of GeDH from the oxidation of geraniol.
An exemplary heterologous or endogenous pathway in yeast and bacterial for the production of d- or l-citronellal and d- or l-citronellol is shown in
An exemplary heterologous or endogenous pathway in yeast and bacterial for the production of d- or l-citronellal and d- or l-citronellol is shown in
In vivo expression of heterologous genes that establish the citronellal/citronellol pathway on plasmid in S. cerevisiae was tested using a yeast strain with elevated levels of isopentenyl diphosphate (IPP) and dimethylally pyrophosphate (DMAPP), caused by a transcriptional downregulation of ERG20 to increase the production of geranyl diphosphate by the overexpression of Ag_GPPS2 (see e.g., WO 2014027118 which is incorporated by reference in its entirety).
The yeast strain was transformed with plasmids containing autonomously replicating sequence (ARS) and a yeast centromere (CEN) (ARS-CEN plasmids) with co-expression of Abies grandis geranyl diphosphate synthase (Ag_GPPS2), Catharanthus roseus geranial synthase (Cr_GES), and/or Kluyveromyces lactis Yellow Enzyme (KI_KYE1) and Castellaniella defragrans geranial dehydrogenase (Cd_GeDH). The ARS-CEN plasmid was under the control of constitutive promoters. Synthetic Complete (SC) media with 2% glucose was used for culturing. Cultures were supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 120 hours.
All genes listed above were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants and IPM samples were analyzed by UPCs-UV.
Co-expression of Ag_GPPS2+Cr_GES resulted in production of about 400 mg/L of geraniol, the first intermediate in the pathway. Co-expression of Ag_GPPS2+Cr_GES+KI_KYE1 led to the production about 300 mg/L of geraniol and a minor accumulation of geranial (about 10 mg/L) and citronellol (about 25 mg/L). Expression of Ag_GPPS2+Cr_GES+Cd_GeDH led to accumulation of varying amounts of geraniol (about 30 mg/L), geranial (about 150 mg/L), citronellol (about 100 mg/L), neral (about 75 mg/L), and less than 5 mg/L of citronellal and nerol. These results demonstrate that geraniol is converted to geranial by Cd_GeDH and then further converted transiently to citronellal by endogenous ene reductase activity (Sc_OYE2). Some of the geranial is interconverted to neral by keto-enol tautomerisation and some of the neral is reduced to nerol by AR activity. Following the production of citronellal, alcohol dehydrogenase/aldehyde reductase activity yields citronellol. Expression of Ag_GPPS2, Cr_GES, Cd_GeDH and KI_KYE1 led to the establishment of the full citronellal/citronellol pathway with production of citronellol (via citronellal). Expression of KI_KYE1 and Cd_GeDH genes on the plasmid suggest their role in the production of intermediates and/or the end product of the citronellal/citronellol pathway. The predominant direction of the pathway was revealed by the stepwise addition of pathway steps. GPP→geraniol/nerol→geranial/neral→citronellal→citronellol (see
In vivo expression of heterologous genes that establish the production of nerol via activation of the citronellal/citronellol pathway on plasmids in S. cerevisiae was tested using a yeast strain with elevated levels of isopentenyl diphosphate (IPP) and dimethylally pyrophosphate (DMAPP), caused by a transcriptional downregulation of ERG20.
The yeast strain was transformed with plasmids containing autonomously replicating sequence (ARS) and a yeast centromere (CEN) (ARS-CEN plasmids) with co-expression of Abies grandis_geranyl diphosphate synthase (Ag_GPPS2) and Glycine max_nerol synthase (Gm_NES; SEQ ID NO:56) or Solanum lycopersicum_neryl diphosphate synthase (SI_NDPS1; SEQ ID NO:53) and Gm_NES. The ARS-CEN plasmid was under the control of constitutive promoters. Synthetic Complete (SC) media with 2% glucose was used for culturing. Cultures were supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 120 hours.
All genes listed above were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants and IPM samples were analyzed by UPCs-UV. Expression of Ag_GPPS2 and Gm_NES resulted in the production of geraniol only (about 30 mg/L) while the expression of SI_NDPS1 and Gm_NES yielded about 5 mg/L of nerol and geraniol (see
In vivo expression of heterologous genes that establish the citronellal/citronellol pathway on plasmids in S. cerevisiae was tested using a yeast strain with elevated levels of isopentenyl diphosphate (IPP) and dimethylally pyrophosphate (DMAPP), caused by a transcriptional downregulation of ERG20.
The yeast strain was transformed with plasmids containing autonomously replicating sequence (ARS) and a yeast centromere (CEN) (ARS-CEN plasmids) with co-expression of Abies grandis geranyl diphosphate synthase (Ag_GPPS2) and Catharanthus roseus geranial synthase (Cr_GES), co-expression of Ag_GPPS2, Cr_GES, and Olea europaea Iridoid synthase (Oe_ISY), or co-expression of Ag_GPPS2, Cr_GES, Oe_ISY, and Bradyrhizobium sp. DFCI-1_citronellol/citronellal dehydrogenase (Bs_CiDH). The ARS-CEN plasmid was under the control of constitutive promoters. Synthetic Complete (SC) media with 2% glucose was used for culturing. Cultures were supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 120 hours.
All genes listed above were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants and IPM samples were analyzed by UPCs-UV.
Expression of Ag_GPPS2 and Cr_GES resulted in the production of about 230 mg/L of geraniol. Expression of Ag_GPPS2, Cr_GES and Oe_ISY lead to the production of about 200 mg/L geraniol, about 50 mg/L citronellol and less than 10 mg/L citronellal. Expression of Ag_GPPS2, Cr_GES, Oe_ISY, and Bs_CiDH yielded 375 mg/L geraniol, 100 mg/L citronellol, less than 50 mg/L citronellal, and less than 20 mg/L geranial. Results indicate that Oe_ISY converts geraniol directly to citronellol and that co-expression of Bs_CiDH in a yeast strain with geraniol leads to the accumulation of a small amount of citronellal (see
S. cerevisiae yeast strain with full chromosomal integration of citronellal/citronellol pathway genes Abies grandis geranyl diphosphate synthase (Ag_tGPPS) Catharanthus roseus geranial synthase (Cr_GES) Kluyveromyces lactis Yellow Enzyme (KI_KYE1), and Castellaniella defragrans geranial dehydrogenase (Cd_GeDH) was developed and tested to identify which citronellal/citronellol pathway intermediates and/or end product would be produced.
A yeast strain with elevated levels of isopentenyl diphosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP), caused by transcriptional downregulation of ERG20, was used for integration of pathway expression cassettes (see e.g., WO 2014027118 which is incorporated by reference in its entirety). TEF1 promoter in front of Ag_GPPS2 PGK1 promoter in front of Cr_GES and Cd_GeDH, and TPI1 promoter in front of KI_KYE1. The expression cassettes containing flanking regions were integrated in the yeast genome by homologous recombination The integration construct consists of an expression cassette and a selection marker (promoter-ORF-terminator-selection marker) with flanking sequences upstream and downstream (about 3-400 nucleotides) that are homologous to specific intergenic sequences in the S. cerevisiae genome. The homologous sequences target the integration construct to these sequences in the genome, and the construct integrates by homologous recombination. Yeast clones with integrated expression cassette and selection marker can be selected by means of selection marker.
Yeast extract Peptone Dextrose (YPD) media with 2% glucose was used for culturing. The cultures were supplemented with 10% v/v isopropyl myristate (IPM) secondary phase during culturing to help extract and trap molecules produced by the activation of the pathway. Cultures were grown for 120 hours. All genes were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants. IPM samples were analyzed by UPC2-UV. IPM samples were analysed by chiral GC to measure optical purity of citronellal and citronellol (enantiomer excess; ee %).
Expression of the integrated pathway resulted in the production of citronellol (about 175 mg/L) as the only major product. Specifically, the integrated pathway yielded about 100 ee % of d-citronellol (see
The active geraniol dehydrogenases in the citronellal/citronellol pathway in yeast include Castellaniella defragrans geraniol dehydrogenase (Cd_GeDH), Thauera terpenica 58Eu geraniol dehydrogenase (Tt_GeDH), Rhodococcus sp. RD6.2 geraniol dehydrogenase (Rs_GeDH), Spingopyxis macrogoltabida geraniol dehydrogenase (Sm_GeDH), Acinetobacter calcoaceticus geraniol dehydrogenase (Ac_GeDH), and Pseudomonas putida geraniol dehydrogenase (Pp_GeDH).
In order to initiate the citronellal/citronellol pathway, a yeast strain with elevated levels of IPP and DMAPP, caused by a transcriptional downregulation of ERG20 was used to yield elevated levels of GPP. The yeast strain was transformed with ARS-CEN plasmids with Abies grandis geranyl diphosphate synthase (Ag_GPPS2), Catharanthus roseus geranial synthase (Cr_GES), and Kluyveromyces lactis Yellow Enzyme (KI_KYE1), and a geraniol dehydrogenase gene selected from the following: Castellaniella defragrans geraniol dehydrogenase (Cd_GeDH; SEQ ID NO:1), Thauera terpenica 58Eu geraniol dehydrogenase (Tt_GeDH; SEQ ID NO:5), Rhodococcus sp. RD6.2_geraniol dehydrogenase (Rs_GeDH; SEQ ID NO:2), Spingopyxis macrogoltabida geraniol dehydrogenase (Sm_GeDH; SEQ ID NO:3), and Acinetobacter calcoaceticus geraniol dehydrogenase (Ac_GeDH; SEQ ID NO:4), Pseudomonas putida geraniol dehydrogenase (Pp_GeDH; SEQ ID NO:6). The heterologous genes are under control of constitutive promoters; TEF1 promoter in front of Ag_GPPS2, PGK1 promoter in front of Cr_GES, TPI1 promoter in front of KI_KYE1 and PGK1 promoter in front of each of the different GeDHs. Synthetic Complete (SC) media with 2% glucose was used for culturing. Cultures were supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules coming from the pathway. Cultures were grown for 120 hours. All genes were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants and IPM samples were analyzed by UPCs-UV.
Various combinations of Cd_GeDH, Tt_GeDH, Rs_GeDH, Sm_GeDH, Pp_GeDH, and Ac_GeDH enzymes tested in this experiment yielded various amounts of citronellol (see
Citronellal/citronellol pathway production was carried out in yeast that overexpressed endogenous ENR or Sc_OYE2 (plasmid-based). The yeast strain was transformed with plasmids containing autonomously replicating sequence (ARS) and a yeast centromere (CEN) (ARS-CEN plasmids) with co-expression of Abies grandis geranyl diphosphate synthase (Ag_GPPS2), Catharanthus roseus geranial synthase (Cr_GES), and Castellaniella defragrans geranial dehydrogenase (Cd_GeDH)(control) or Ag_GPPS2, Cr_GES, Cd_GeDH, and S. cerevisiae ENR (Sc_OYE2; SEQ ID NO:33). The ARS-CEN plasmid was under the control of constitutive promoters. Synthetic Complete (SC) media with 2% glucose was used for culturing. Cultures were supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 120 hours.
All genes listed above were synthesized by Thermo Fisher Scientific GENEART GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants and IPM samples were analyzed by UPCs-UV. IPM samples were analyzed by chiral GC to measure optical purity of citronellal and citronellol.
The expression of Ag_tGGPS, Cr_GES and Cd_GeDH yielded about 10 mg/L geraniol, less than 5 mg/L nerol, about 30 mg/L geranial, about 10 mg/L neral, about 45 mg/L citronellol, and less than 5 mg/L citronellal. Endogenous ENR activity resulted in the production of 96.4% ee d-citronellol. The overexpression of Sc_OYE2 produced about 10 mg/L geraniol, less than 5 mg/L nerol, about 25 mg/L geranial, about 10 mg/L neral, about 50 mg/L citronellol, and less than 5 mg/L citronellal. D-citronellol with an ee of 97.0% was produced (see
A variety of GeDH genes were expressed in yeast cultures that were used for preparing yeast cell lysate. The lysate was used for feeding experiments with geraniol to observe the amount of citronellal/citronellol pathway products produced.
Yeast cell lysates (YCL) from yeast cultures expressing Cd_GeDH, Tt_GeDH, Rs_GeDH, Sm_GeDH, Pp_GeDH, or Ac_GeDH were harvested and diluted to similar protein concentrations. Following harvesting, in vitro reactions were initiated by feeding geraniol and NAD as substrates to the lysates to establish the citronellal/citronellol pathway. To measure background activity that was not a result of the expression of GeDH, the YCLs were harvested from cells only containing an empty vector (see “Empty”). In vitro reactions were stopped by extracting with Methyl-tert-butylether (MTBE) after 1, 15, and 90 minutes of incubation at 30° C. and levels of geranial, citronellal and citronellol were analyzed by gas chromatography-flame ionization detector (GC-FID). All genes were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants.
All YCLs harvested from cells expressing a GeDH showed higher signals of geranial, citronellal, and citronellol than YCL harvested from cells expressing the empty vector (compare “X_GeDH” vs. “Empty”) (see
Overexpression of ENRs KI_KYE1 (SEQ ID NO:7) and Zymomonas mobilis ENR (Zm_OYE; SEQ ID NO:9) was carried out in yeast produced citronellol of high purity. The yeast strain was transformed with plasmids containing autonomously replicating sequence (ARS) and a yeast centromere (CEN) (ARS-CEN plasmids) with co-expression of Abies grandis geranyl diphosphate synthase (Ag_GPPS2), Catharanthus roseus geranial synthase (Cr_GES), and Castellaniella defragrans geranial dehydrogenase (Cd_GeDH) (control) or Ag_GPPS2, Cr_GES, Cd_GeDH, and, Cd_GeDH, and Zm_OYE. The ARS-CEN plasmid was under the control of constitutive promoters. Synthetic Complete (SC) media with 2% glucose was used for culturing. Cultures were supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 120 hours.
All genes listed above were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants and IPM samples were analyzed by UPCs-UV. IPM samples were analyzed by chiral GC to measure optical purity of citronellal and citronellol.
Expression of the control genes in the plasmid-based citronellal/citronellal production pathway in yeast yielded less than 5 mg/L nerol, about 20 mg/L geranial, about 10 mg/L neral, and about 100 mg/L citronellol. Overexpression of KI_KYE1 resulted in about 5 mg/L neral and about 200 mg/L d-citronellol (97.6% ee). Overexpression of Zm_OYE also produced about 5 mg/L neral and about 200 mg/L l-citronellol (97% ee) (see
GeDHs described in
Citronellal to citronellol conversion was detected in all in vitro reactions (see
In vitro testing of substrate specificity of two different ENRs, KI_KYE1 (SEQ ID NO:21) and Zm_OYE (SEQ ID NO:9), yielded citronellal/citronellol pathway products (see
In vitro incubation of citral (50% geranial:50% neral) with KI_KYE1 alone yielded about 50% geranial and about 50% neral, while 20 hrs of citral incubation with KI_KYE1 overexpression yielded about 30% geranial, about 50% neral, and about 20% citronellal. Primarily, the geranial component is converted to citronellal when KI_KYE is expressed. Under control conditions (without KI_KYE1), without citral incubation, about 50% geranial, and about 50% neral was produced and at 20 hours following citral incubation, about 45% geranial was produced and about 55% neral was produced. The control reaction without KI_KYE was largely unchanged.
In vitro incubation of citral (50% geranial:50% neral) with Zm_OYE alone yielded about 50% geranial, about 40% neral, and about 10% citronellal, while 2 hr incubation yielded about 45% geranial, about 10% neral, and about 45% citronellal. Primarily, the neral component is converted to citronellal. Under control conditions (without Zm_OYE), without citral incubation, and following 2 hours of citral incubation, yielded about 50% geranial, about 45% neral, and about 5% citronellal. The control reaction without Zm_OYE was largely unchanged.
A mevalonate plasmid for the expression of IPP and DMAPP in E. coli was constructed. Seven genes (Ec_atoB (SEQ ID NO:10), Sa_mvaS (SEQ ID NO:11), Sa_mvaA (SEQ ID NO:12), Sc_Erg12 (SEQ ID NO:13), Sc_erg8 (SEQ ID NO:14), Sc_erg19 (SEQ ID NO:15), and Ec_idi (SEQ ID NO:16)) were subdivided on two operons. The first three genes were placed under one promoter and the last four genes were placed under another promoter. Each operon was engineered to contain a transcriptional terminator. The 7 genes are located on a p15-based replicative plasmid backbone encoding the LacI protein and a kanamycin selection marker. All genes except Ec_idi were heterologous and were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as Escherichia coli codon optimized variants (see
A plasmid comprising genes for citronellal production from IPP and DMAPP in E. coli was constructed. Four proteins (geranyl diphosphate synthase, geraniol synthase, ene reductase and geraniol dehydrogenase) encoded by Ag_GPPS2, Cr_GES, KI_KYE1, and Cd_GeDH (alternatively another GPPS, GES, GeDH, or ENR gene as disclosed throughout) were subdivided on two operons. The first three genes were under the control of one promoter and the last gene is under the control of another promoter. The 4 genes were located on a pBR322-based replicative plasmid backbone encoding ampicillin selection marker. All genes were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as E. coli codon optimized variants, except Rs_GeDH which was codon optimized for Saccharomyces cerevisiae (see
Plasmid constructs from
Plasmid pMev encoded 7 proteins (Ec_atoB, Sa_mvaS, Sa_mvaA, Sc Mk, Sc_erg8, Sc_erg19, and Ec_idi) responsible for the conversion of acetyl-CoA to IPP and DMAPP and plasmid pCitro encoded 4 proteins (Ag_GPPS2, Cr_GES, KI_KYE1 and Rs_GeDH or Cd_GeDH) that convert IPP and DMAPP to citronellal/citronellol pathway products. Cultures were grown for 40 hours in LB media containing 1% glucose. Cultures were supplemented with 10% IPM secondary phase to help extract and trap the pathway intermediates. IPM samples were analyzed in triplicates by UltraPerformance Convergence Chromatography (UPC2-UV) (triplicates). IPM samples were analyzed by chiral GC to measure optical purity (enantiomer excess; ee %) of citronellal and citronellol.
Co-expression of pMev and either pCitro (Cd_GeDH) or pCitro (Rs_GeDH) in E. coli resulted in the production of citronellal/citronellol intermediates and citronellol. About 25 mg/L of geranial, about 5 mg/L nerol, about 350 mg/L d-citronellol (94% ee), and about 40 mg/L d-citronellal (93.4% ee) was produced with the co-expression of pMEV and pCitro (Cd_GeDH+KI_KYE1). When Cd_GeDH was replaced by Rs_GeDH, about 75 mg/L geraniol, about 25 mg/L geranial, about 5 mg/L nerol, about 40 mg/L d-citronellol (95% ee), and about 300 mg/L d-citronellal (94.2% ee) (see
Determination of citronellal/citronellol pathway production with overexpression of Zm_OYE on plasmid in E. coli.
Plasmid pMev encodes seven genes (Ec_atoB, Sa_mvaS, Sa_mvaA, Sc_MK, Sc_erg8, Sc_erg19, and Ec_idi) which convert acetyl-CoA to IPP and DMAPP and plasmid pCitro encodes four genes; Ag_GPPS2, Cr_GES, Zm_OYE) and Cd_GeDH which convert IPP and DMAPP to citronellal/citronellol. Cultures were grown for 40 hours in LB media containing 1% glucose and supplemented with 10% IPM secondary phase to help extract and trap the pathway intermediates and end product. All pathway products were analyzed in triplicates by UPC2-UV. IPM samples were analyzed by chiral GC to measure optical purity (enantiomer excess (ee %)) of citronellal and citronellol. Co-expression of plasmids pMev and pCitro (Cd_GeDH+Zm_OYE) resulted in the production of about 175 mg/L l-citronellol (99.6% ee) and about 150 mg/L l-citronellal (99.4% ee) (see
Endogenous yeast genes that directly or indirectly are involved in aldehyde reductase (AR) activity were deleted in a yeast strain that converts citronellal to citronellol in order to control the amount of citronellal and citronellol produced by the pathway in the yeast. This approach can lead to an increase in the chemical purity of citronellal produced in yeast.
An S. cerevisiae yeast strain with elevated levels of IPP and DMAPP (caused by a transcriptional downregulation of ERG20), was used for integration of citronellal/citronellol pathway expression cassettes Ag_GPPS, Cr_GES, Cd_GeDH, KI_KYE1 under control of constitutive promoters (all genes were codon-optimized for expression in S. cerevisiae using GENEART™, and expression cassettes were integrated in the yeast genome by homologous recombination). Deletion of combinations of the following yeast genes: ADH6, RFX1, GRE2, ARI1, GCY1 and AYR1, led to an increase in citronellal accumulation. The yeast strains were grown for 96 hours in synthetic complete (SC) media with 2% glucose, supplemented with 10% v/v isopropylmyristate (IPM) secondary phase during culture to promote extraction and trapping of the targeted citronellal/citronellol pathway molecules. IPM samples were analyzed by UPC2-UV and by chiral GC (see
As shown in
Expression of a heterologous NADH oxidase gene in a yeast strain comprising the citronellal/citronellol pathway reduces citronellol formation. NADH oxidase converts NADH to NAD+, thus lowering NADH levels in the yeast. The lower NADH levels lead to reduced enzymatic activity of aldehyde reductase (AR), and thus less conversion of citronellal to citronellol in yeast. This approach can increase the chemical purity of citronellal produced in yeast by reducing citronellol accumulation.
An S. cerevisiae yeast strain with elevated levels of IPP and DMAPP (caused by a transcriptional downregulation of ERG20), was used for integration of citronellal/citronellol pathway expression cassettes Ag_GPPS, Cr_GES, Rs_GeDH, KI_KYE1 under control of constitutive promoters (all genes were codon-optimized for expression in S. cerevisiae using GENEART™, and expression cassettes were integrated in the yeast genome by homologous recombination). Expression of Sp_NADHoxi (SEQ ID NO:69), led to reduced accumulation of citronellol and increased chemical purity of citronellal. The yeast strains were grown for 96 hours in synthetic complete (SC) media with 2% glucose, supplemented with 10% v/v isopropylmyristate (IPM) secondary phase during culture to promote extraction and trapping of the targeted citronellal/citronellol pathway molecules. IPM samples were analyzed by UPC2-UV and by chiral GC (see
As shown in
Expression of a heterologous carboxylic acid reductase (CAR) gene together with a heterologous phosphopantetheine transferase (PPTase) gene in a yeast strain comprising the citronellal/citronellol pathway reduces citronellic acid accumulation. Expression of a carboxylic acid gene together with phosphopantetheine transferase gene in yeast with citronellal/citronellol pathway can prevent carboxylic acid formation. Expression of a CAR gene and PPTase gene prevents citronellal from being converted to citronellic acid when citronellal accumulates in yeast upon deletion of genes that support aldehyde reductase (AR) activity. Thus, this approach can be used as a means to increase chemical purity of citronellal in yeast.
An S. cerevisiae yeast strain with elevated levels of IPP and DMAPP (caused by a transcriptional downregulation of ERG20), was used for integration of citronellal/citronellol pathway expression cassettes Ag_GPPS, Cr_GES, Rs_GeDH, KI_KYE1 under control of constitutive promoters (all genes were codon-optimized for expression in S. cerevisiae using GENEART™, and expression cassettes were integrated in the yeast genome by homologous recombination). Expression of Mm_CAR (SEQ ID NO:70) and Bs_SFP (SEQ ID NO:71), led to reduced accumulation of citronellol and increased chemical purity of citronellal. The yeast strains were grown for 96 hours in synthetic complete (SC) media with 2% glucose, supplemented with 10% v/v isopropylmyristate (IPM) secondary phase during culture to promote extraction and trapping of the targeted citronellal/citronellol pathway molecules. IPM samples were analyzed by UPC2-UV and by chiral GC (see
As shown in
In vivo expression of heterologous genes that establish a citronellal/citronellol pathway on plasmid was tested in yeast using a S. cerevisiae strain with elevated levels of isopentenyl diphosphate (IPP) and dimethylally pyrophosphate (DMAPP), caused by a transcriptional downregulation of ERG20. The yeast strain was further transformed with plasmids expressing Rhodococcus sp. geranial dehydrogenase (Rs_GeDH; SEQ ID NO:2) or Castellaniella defragrans geranial dehydrogenase (Cd_GeDH; SEQ ID NO:1) and/or Kluyveromyces lactis_Yellow Enzyme (KI_KYE1; SEQ ID NO:7), using constitutive promoters, to establish the citronellal/citronellol pathway. All genes listed above were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants. Synthetic Complete (SC) media with 2% glucose and supplemented with 250 mg/L nerol in IPM was used for culturing. Cultures were also supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 96 hours and IPM samples were analyzed by UPCs-UV.
Feeding of nerol to yeast expressing heterologous genes with Ne/GeDH activity and ene reductase activities leads to production of neral, d- and l-citronellal as well as d- and l-citronellol. In a strain containing the geraniol dehydrogenase Cd_GeDH (or Rs_GeDH or others), nerol is converted to neral and then to citronellol via citronellal (being converted to citronellol by ADH background activities). In a strain containing both a geraniol dehydrogenase and the ene reductase KI_KYE, all the nerol is converted to citronellol via citronellal (being converted to citronellol by ADH background activities). This approach shows that nerol can be converted to citronellal and citronellol by heterologous genes with Ne/GeDH and ene-reductase activities, and this approach can be used as an alternative to the main pathway, as described in example 7 above, to produce d- and l-citronellal d- and l-citronellol (see
In vivo expression of heterologous genes that establish a citronellal/citronellol pathway on plasmid was tested in yeast using a S. cerevisiae strain with elevated levels of isopentenyl diphosphate (IPP) and dimethylally pyrophosphate (DMAPP), caused by a transcriptional downregulation of ERG20. The yeast strain was transformed with plasmids expressing an Iridoid synthase (Oe_ISY) (SEQ ID NO:54) and/or a ene reductase (KI_KYE1; SEQ ID NO:7) under control of constitutive promoters. All genes listed above were synthesized by Thermo Fisher Scientific GENEART™ GmbH (Regensburg, Germany) as S. cerevisiae codon optimized variants. Synthetic Complete (SC-His) media with 2% glucose and supplemented with 200 mg/L geraniol or 250 mg/L nerol in IPM was used for culturing. Cultures were also supplemented with 10% v/v isopropylmyristate secondary phase during the culturing to help extract and trap targeted molecules produced by the activation of the pathway. Cultures were grown for 96 hours and IPM samples were analyzed by UPCs-UV.
Feeding of geraniol or nerol to yeast expressing heterologous genes with ene reductase activity (ENR or Iridoid synthase ISY) leads to direct conversion to d- and l-citronellol, respectively. In a strain containing Oe_ISY, geraniol and nerol are converted to citronellol. This approach show that geraniol and nerol can be converted to citronellol by a heterologous a Iridoid synthase gene. This approach can be used as an alternative to the main pathway, as described in example 7 above, to produce d- and l-citronellal, and d- and l-citronellol (see
Citronellol produced by recombinant microorganisms (“strains from NCCB”) were contacted with the oxidizing bacteria Gluconobacter oxydans or Gluconobacter cerinus to perform the bioconversion of citronellol into citronellal. The oxidizing bacteria were cultured in a glycerol medium with 2.5% glycerol, 0.5% yeast extract, and 0.3% peptone. Cultures were harvested and resuspended in 50 mM acetate or phosphate buffer (pH 5 or 6.5), and supplemented with 10% v/v isopropylmyristate secondary phase containing 0.5 g/L citronellol to decrease toxicity of the substrate and the product citronellal or citronellic acid (in some cases, 2.5% glycerol was added to the bioconversion). Bioconversion was performed for 24 to 144 hours. IPM samples were analyzed by UPCs-UV to detect products.
The example identifies that the oxidizing activity of the oxidizing bacteria Gluconobacter oxydans and Gluconobacter cerinus converts citronellol produced by recombinant microorganisms into citronellal or citronellic acid, and demonstrates an approach of bioconversion of citronellol into citronellal. The oxidizing bacteria Gluconobacter oxydans produced about 100 mg/L of citronellal, and about 200 mg/L of citronellic acid after 144 hours (see
Citronellal produced by recombinant microorganisms (“strains from NCCB”) were contacted with the oxidizing bacteria Gluconobacter cerinus to perform the bioconversion of citronellal to citronellic acid. The oxidizing bacteria were cultured in a glycerol medium with 2.5% glycerol, 0.5% yeast extract, and 0.3% peptone. Cultures were harvested and resuspended in 50 mM acetate (pH 5), and supplemented with 10% v/v isopropylmyristate secondary phase containing 0.5 g/L citronellal to decrease toxicity of the substrate and the product. Bioconversion was performed for 144 hours. IPM samples were analyzed by UPCs-UV to detect products.
The example identifies that the oxidizing activity of the oxidizing bacteria Gluconobacter cerinus converts citronellal produced by recombinant microorganisms into citronellic acid, and demonstrates an approach of bioconversion of citronellal into citronellic acid. The oxidizing bacteria Gluconobacter cerinus converted 100% of the citronellal into citronellic acid after 144 hours (see
Citronellol produced by recombinant microorganisms (“strains from NCCB”) were contacted with the oxidizing bacteria Gluconobacter cerinus or Gluconobacter frateurii to perform the bioconversion of citronellol to citronellic acid. The oxidizing bacteria were cultured in a glycerol medium with 2.5% glycerol, 0.5% yeast extract, and 0.3% peptone. Cultures were harvested and resuspended in 50 mM acetate (pH 5), and supplemented with 10% v/v isopropylmyristate secondary phase containing 0.5 g/L citronellol to decrease toxicity of the substrate and the product. Bioconversion was performed for 144 hours. IPM samples were analyzed by UPCs-UV to detect products.
The example identifies that the oxidizing activity of the oxidizing bacteria Gluconobacter cerinus or Gluconobacter frateurii converts citronellol produced by recombinant microorganisms into citronellic acid, and demonstrates an approach of bioconversion of citronellol into citronellic acid. The oxidizing bacteria Gluconobacter cerinus or Gluconobacter frateurii converted 100% of the citronellol into citronellic acid after 144 hours (see
This application is a U.S. National Stage Application of International Application No. PCT/EP2017/075995, filed on Oct. 11, 2017, and claims the benefit of U.S. Provisional Application No. 62/406,906, filed on Oct. 11, 2016, the disclosures of each of which are explicitly incorporated by reference herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
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| PCT/EP2017/075995 | 10/11/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/069418 | 4/19/2018 | WO | A |
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| 20100112671 | Keasling et al. | May 2010 | A1 |
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| WO 2014027118 | Feb 2014 | WO |
| WO 2016008883 | Jan 2016 | WO |
| WO-2016108236 | Jul 2016 | WO |
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| Number | Date | Country | |
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| 20190225945 A1 | Jul 2019 | US |
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| 62406906 | Oct 2016 | US |
