Patent Grant
12098397
The contents of the electronic sequence listing (“338451_71-20A.xml”; Size; 134,736 bytes; and Date of Creation: Aug. 2, 2022) is herein incorporated by reference in its entirety.
The present invention relates to materials and methods of producing loline alkaloids or precursors thereof.
Loline alkaloids are produced symbiotically during infection of grasses by Epichloë species fungal endophytes. These endophytes are considered to be bioprotective, conferring pest, and possibly drought and disease protection to the symbionts of which they form part.
Lolines are potent broad spectrum insecticidal alkaloids with no observed toxicity to animals. These fungal secondary metabolites are major contributors to the bioprotective pest tolerance conferred on cool season grasses by Epichloë endopytes.
A robust and/or scalable method for preparing loline alkaloids in the absence of a symbiotic relationship is not presently available.
There is a need for a method to produce loline alkaloids in fungi that do not natively produce lolines in order to extract and use lolines as a natural pesticide.
It is an object of the present invention to provide improved materials and methods for producing loline alkaloids or precursors thereof, and/or at least provide the public with a useful choice.
Host Cell
In one aspect, the invention provides a host cell modified or transformed to comprise at least one polynucleotide selected from the group consisting of:
In one embodiment the host cell is modified or transformed to comprise at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11 polynucleotides selected from i) to xi)
In a further embodiment the host cell is modified or transformed to comprise the polynucleotides of i), ii), iii), v) and vii). That is the host cell is modified or transformed to comprise a lolC, lolF, lolD, lolT and a lolO gene, or polynucleotides encoding a lolC, lolF, lolD, lolT and a lolO gene product.
In a further embodiment the host cell contains more copies of the at least one polynucleotide than does a control cell.
In a further embodiment the host cell is not modified or transformed to comprise the polynucleotide of vi). That is the host cell is not modified or transformed to comprise a lolE gene, or a polynucleotide encoding a lolE gene product.
In a further embodiment the host cell is not modified or transformed to comprise the polynucleotide of viii). That is the host cell is not modified or transformed to comprise a lolU gene, or a polynucleotide encoding a lolU gene product.
In a further embodiment the host cell is not modified or transformed to comprise the polynucleotides of vi) or viii). That is the host cell is not modified or transformed to comprise a lolE or a lolU gene, or polynucleotides encoding a lolE or a lolU gene product.
In one embodiment the host cell produces more of at least one loline alkaloid or precursor thereof, than does a control cell.
In a further embodiment the host cell produces more of at least one loline alkaloid or precursor thereof, than does a control cell, as a result of the host cell being transformed or modified to comprise at least one polynucleotide.
In a further embodiment the control cell has not been modified or transformed to comprise the at least one polynucleotide.
In a further embodiment the control cell is of the same species or strain as the host cell that has been modified or transformed to comprise the at least one polynucleotide
Host Cell where Polynucleotide is Part of an Expression Construct
In a further embodiment the at least one polynucleotide is part of an expression construct.
In a further embodiment the at least one polynucleotide is operably linked to a promoter.
In a further embodiment the at least one polynucleotide is operably linked to a terminator.
In a further embodiment the at least one polynucleotide is operably linked to a promoter and a terminator.
Expression Construct
In a further aspect, the invention provides an expression construct comprising at least one polynucleotide selected from the group consisting of:
In one embodiment the expression comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11 polynucleotides selected from i) to xi).
In a further embodiment the expression construct comprises at least the polynucleotides of i), ii), iii), v) and vii). That is the construct comprises at least a lolC, lolF, lolD, lolT and a lolO gene, or at least polynucleotides encoding a lolC, lolF, lolD, lolT and a lolO gene product.
In a further embodiment the at least one polynucleotide is operably linked to at least one promoter.
In a further embodiment the expression construct does not comprise the polynucleotide of vi). That is the expression construct does not comprise a lolE gene, or a polynucleotide encoding a lolE gene product.
In a further embodiment the expression construct does not comprise the polynucleotide of viii). That is the expression construct does not comprise a lolU gene, or a polynucleotide encoding a lolU gene product.
In a further embodiment the expression construct does not comprise the polynucleotides of vi) or viii). That is the expression construct does not comprise a lolE or a lolU gene, or polynucleotides encoding a lolE or a lolU gene product.
Host Cell Comprising the Construct
In a further aspect the invention provides a host cell comprising at least one construct of the invention.
Those skilled in the art will understand that the desired complement of lol genes or polynucleotides may be present in one or multiple constructs that are transformed into the host.
In a preferred embodiment the at least one polynucleotide, or expression construct, is stably incorporated into the genome of the host cell.
Host Cell is Tolerant of ACPP Production
In a further embodiment the host cell is tolerant of endogenous (3-amino-3-carboxypropyl)proline (ACPP) production.
In a further embodiment the host cell has been pre-selected for tolerance to said level of cellular ACPP.
In a further embodiment the host cell has been pre-selected for tolerance of endogenous ACPP production by transformation with at least one polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:1 or a variant thereof with at least 40% identity to SEQ ID NO:1 with at least one of activity of a gamma-class PLP enzyme and activity substantially equivalent to that of a lolC gene product.
In a further embodiment the host cell was supplied, or fed, with O-acetyl-L-homoserine (OAH) during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts of OAH during selection.
In a further embodiment the host cell was supplied, or fed, with L-proline during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts of L-proline during selection.
In a further embodiment the host cell is tolerant to at least 0.2 mM, more preferably at least 0.4 mM, more preferably at least 0.6 mM, more preferably at least 0.8 mM, more preferably at least 1 mM, more preferably at least 1.2 mM, more preferably at least 1.4 mM, more preferably at least 1.6 mM, more preferably at least 1.8 mM, more preferably at least 2 mM, more preferably at least 2.2 mM, more preferably at least 2.4 mM, more preferably at least 2.6 mM, more preferably at least 2.8 mM, more preferably at least 3 mM, more preferably at least 3.2 mM, more preferably at least 3.4 mM, more preferably at least 3.6 mM, more preferably at least 3.8 mM, more preferably at least 4 mM, more preferably at least 4.2 mM, more preferably at least 4.4 mM, more preferably at least 4.6 mM, more preferably at least 4.8 mM, more preferably at least 5 mM, more preferably at least 5.2 mM, more preferably at least 5.4 mM, more preferably at least 5.6 mM, more preferably at least 5.8 mM, more preferably at least 6 mM, more preferably at least 6.2 mM, more preferably at least 6.4 mM, more preferably at least 6.6 mM, more preferably at least 6.8 mM, more preferably at least 7 mM, more preferably at least 7.2 mM, more preferably at least 7.4 mM, more preferably at least 7.6 mM, more preferably at least 7.8 mM, more preferably at least 8 mM ACPP in the growth medium.
In a further embodiment host cell is tolerant of a level of cellular ACPP that is toxic to a control cell of the same strain or species.
Host Cell can Convert 1-AP to AcAP
In a further embodiment the host cell, prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).
In a further embodiment the host cell, prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.
In a further embodiment the host cell has been pre-selected by measuring AcAP production in the host cell.
In a further embodiment the host cell was supplied, or fed, with 1-AP during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts 1-AP during selection.
In a further embodiment the selected host cell can produce at least 0.005 milligrams (mg), preferably 0.01 milligrams (mg), more preferably 0.02 milligrams (mg), more preferably 0.03 milligrams (mg), more preferably 0.04 milligrams (mg), 0.05 milligrams (mg), more preferably at least 0.1 mg, more preferably at least 0.15 mg, more preferably at least 0.2 mg, more preferably at least 0.25 mg, more preferably at least 0.3 mg, more preferably at least 0.35 mg, more preferably at least 0.4 mg, more preferably at least 0.45 mg, more preferably at least 0.5 mg, more preferably at least 0.75 mg, more preferably at least 1 mg, more preferably at least 1.5 mg, more preferably at least 2 mg of AcAP per gram (g) of cellular biomass.
Host Cell Type
In a further embodiment the cell is from a fungal species.
In a further embodiment the cell is from a bacterial species.
In a further embodiment the cell is from the subkingdom Dikarya.
In a further embodiment the cell is from a phylum selected from Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Glomeromycota, Ascomycota and Basidiomycota or a subphylum incertae sedis selected from Mucoromycotina, Kickxellomycotina, Zoopagomycotina and Entomophthoromycotina.
In a further embodiment the cell is from an order selected from Mucorales, Hypocreales, Eurotiales, Sebacinales and Saccharomycetales.
In a further embodiment the cell is from a genus selected from Metarhizium, Epichloë, Saccharomyces, Kluveromyces, Trichoderma, Aspergillus, Beauveria, Pichia, Penicillium, Serendipita, Umbelopsis, Neurospora, Epicoccum, Sarocladium, Balansia, Fusarium, Alternaria, Ustilago, Sebacina, Glomus and Rhizopus.
In a further embodiment the cell is from the species Metarhizium robertsii. In a further embodiment the cell is from the species Trichoderma reesei. In a further embodiment the cell is from the species Aspergillus niger. In a further embodiment the cell is from the species Aspergillus nidulans. In a further embodiment the cell is from the species Aspergillus oryzae. In a further embodiment the cell is from the species Beauveria bassiana. In a further embodiment the cell is from the species Saccharomyces cerevisiae. In a further embodiment the cell is from the species Pichia pastoris. In a further embodiment the cell is from the species Kluveromyces marxianus. In a further embodiment the cell is from the species Epichloë festucae. In a further embodiment the cell is from the species Epichloë typhina. In a further embodiment the cell is from the species Penicillium chrysogenum. In a further embodiment the cell is from the species Penicillium paxilli. In a further embodiment the cell is from the species Penicillium expansum. In a further embodiment the cell is from the species Serendipita indica. In a further embodiment the cell is from the species Umbelopsis isabellina. In a further embodiment the cell is from the species Neurospora crassa. In a further embodiment the cell is from the species Epicoccum italicum. In a further embodiment the cell is from the species Sarocladium zeae. In a further embodiment the cell is from the species Fusarium verticillioides. In a further embodiment the cell is from the species Ustilago maydis.
In one embodiment, the cell is a fungal cell other than a yeast cell.
In one embodiment the cell is a yeast cell.
In a further embodiment the cell is from a non-Epichloë uncinata fungal species.
Fermentation Suitable Host Cells
In a further embodiment the cell is from species or strain of fungi that is tractable to use in fermentation. In a further embodiment the cell is from a species or strain of fungi capable of a specific growth rate (μ h−1) of at least 0.01, preferably 0.02, more preferably at least 0.03, more preferably at least 0.04, more preferably at least 0.05, more preferably at least 0.1, more preferably at least 0.15, more preferably at least 0.2, more preferably at least 0.25, more preferably at least 0.3, more preferably at least 0.35, more preferably at least 0.4, more preferably at least 0.45.
In a one embodiment the host cells suitable for fermentation is from a phylum selected from: Ascomycota and Basidiomycota or subphylum incertae sedis Mucoromycotina. In one embodiment the host cells suitable for fermentation are from a genus selected from: Aspergillus, Beauveria, Epichloë, Neurospora, Epicoccum, Sarocladium, Kluveromyces, Metarhizium. Penicillium, Pichia, Rhizopus, Saccharomyces, Serendipita, Trichoderma, and Umbelopsis.
In a one embodiment the host cells suitable for fermentation is from a species selected from: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Beauveria bassiana, Epichloë festucae, Epichloë typhina, Epicoccum italicum, Metarhizium robertsii, Penicillium expansum, Penicillium chrysogenum, Penicillium paxilli, Saccharomyces cerevisiae, Kluveromyces marxianus, Pichia pastorus, Rhizopus oryrae, Rhizopus stolonifer, Rhizopus microsporus, Serendipita indica, Trichoderma reesei, Neurospora crassa, Sarocladium zeae and Umbelopsis isabellina.
Method for Producing a Host Cell for Producing at Least One Loline Alkaloid, or Precursor Thereof
In a further aspect the invention provides a method for producing a host cell for producing at least one loline alkaloid or precursor thereof, the method comprising modifying or transforming a host cell to comprise at least one polynucleotide as herein described.
In a further embodiment the host cell is produced by transforming a cell to comprise at least one polynucleotide or construct as herein described.
Method for Producing at Least One Loline Alkaloid or a Precursor Thereof
In a further aspect the invention provides a method for producing at least one loline alkaloid or a precursor thereof, the method comprising culturing host cells of the invention, or produced by a method of the invention, under conditions conducive to the production of the at least one loline alkaloid or precursor thereof, by the host cells.
In one embodiment the method further comprises separating, purifying, fractionating or isolating the at least one loline alkaloid or precursor thereof.
In a further embodiment the host cells are cultured in the presence of at least one loline alkaloid precursor.
In various embodiments the method comprises maintaining the host cells in the presence of at least one of.
In one embodiment the method comprises maintaining the host cells, or a culture thereof, at a temperature of from about 15° C. to about 35° C.
In a further embodiment the method comprises maintaining the host cells, or a culture thereof, at a temperature of from about 15° C. to about 40° C.
In one embodiment the method comprises maintaining the host cells, or a culture thereof, at for at least about 1 day, at least about 3 days, at least about 4 days, at least about 7 days or at least about 10 days.
In one embodiment the method comprises maintaining the host cells, or a culture thereof, in a bioreactor.
In one exemplary embodiment the method is a method of producing one or more loline alkaloids, comprising:
In one embodiment the purification or isolation is achieved via filtration and/or column purification.
In one aspect the invention provides a method for conferring the ability to produce a loline alkaloid or a precursor thereof on an organism, the method comprising transforming the organism with an expression construct of the invention.
In one embodiment the organism, prior to transformation, does not produce the loline alkaloid or precursor thereof.
The cell as herein described may be part of an organism. Thus reference to a cell or host cell can be used interchangeably with reference to an organism or host organism.
In one embodiment the loline alkaloid or precursor thereof is toxic to a pest.
In one embodiment the pest is an insect.
The term “insect” includes, but not limited to, aphids, mealybugs, whiteflies, moths, butterflies, psyllids, thrips, stink bugs, rootworms, weevils, leafhoppers and fruit flies, such as Myzus persicae (green peach aphid), Aphis gossypii Glover (melon/cotton aphid), Rhopalosiphum maidis (corn leaf aphid), Aphis glycines Matsumura (soybean aphid). Brevicoryne brassicae (cabbage aphid), Anasa tristis (squash bug); Pseudococcus longispinus (long tailed mealybug). Pseudococcus calceolariae (scarlet mealybug), Pseudococcus viburni (obscure mealybug), Planococcus citri (Citrus mealybug); Trialeurodes vaporariorum (greenhouse whitefly), Bemisia tabaci (silverleaf whitefly); Plutella xylostella (diamondback moth), Citripestis sagittiferella (citrus fruit moth), Helicoverpa armigera (tomato fruitworm or corn earworm). Pectinophora gossypiella (pink lollworm). Phthorimaea operculella (potato tuber moth), Amyelois transitella (Navel orangeworm), Cydia pomonella (codling moth), Cnephasia jactatana (black-lyre leafroller), Epiphyas postvittana (light-brown apple moth), Grapholita molesta (oriental fruit moth), Ostrinia furnacalis (Asian corn borer), Ostrinia nubilalis (European corn borer), Scirpophaga excerptalis (sugarcane top borer) Diatraea saccharalis (sugarcane borer), Chilo plejadellus (rice stalk borer). Earias vitella (spotted lollworm), Earias insulana (spiny lollworm), Spodoptera frugiperda (fall armyworm), Spodoptera litura (tobacco cutworm), Melittia cucurbitae (squash vine borer), Teia anartoides (painted apple moth), Trichoplusia ni (Cabbage looper); Pieris rapae (white butterfly); Bactericera cockerelli (tomato/potato psyllid), Diaphorina citri (Asian citrus psyllid), Trioza erytreae (African citrus psyllid); Thrips obscuratus (flower thrips), Heliothrips haemorrhoidalis (greenhouse thrips), Thrips tabaci (onion thrips), Frankliniella williamsi (Maize thrip); Halyomorpha halys (brown marmorated stink bug), Oebalus pugnax (rice stink bug), Diabrotica virgifera virgifera (westem corn rootworm), Diabrotica barberi (northern corn rootworm), Diabrotica undecimpunctata howardi (southern corn rootworm), Diabrotica virgifera zeae (Mexican corn rootworm); Pempheres affinis (cotton stem weevil); Nephotettix virescens (green leafhopper), Nilaparvata lugens (brown planthopper); Bactrocera tryoni (Queensland fruit fly).
In one embodiment the pest is a non-insect pest.
In one embodiment the pest is a nematode.
The term “nematode” includes but is not limited to root-knot nematodes (Meloidogyne species), cyst nematodes (Heterodera and Globodera species), lesion nematodes (Pratylenchus species), reniform nematodes (Rotylenchulus reniformis), lance nematodes (Hoplolaimus species) and stem and bulb nematodes (Ditylenchus species).
Preferred LOL genes for use in various aspects and embodiments of the invention are lolC, lolD, lolF, lolT and lolO.
In certain embodiments the host cells and organisms are not transformed to express lolF. In certain embodiments the host cells and organisms and are not transformed to express lolU. In certain embodiments the host cells and organisms are not transformed to express lolE or lolU.
Any one or more of the following embodiments may relate to any of the aspects described herein or any combination thereof.
In will be appreciated that the polynucleotide may be an allelic variant, degenerate sequence, homologue or orthologue of the specified nucleotide sequences.
In various embodiments the variant polypeptide has at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% amino acid identity with the specified polypeptide sequences.
In various embodiments the polynucleotide variant comprises one or more alternative codons that code for the eventual translation of a polypeptide having at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% amino acid identity with the specified polypeptide sequences.
In various embodiments the polynucleotide variant having at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% nucleotide sequence identity to the specified polynucleotide sequence.
Polynucleotide and polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs for e.g., the Needleman-Wunsch global alignment program (Needleman and Wunsch, 1970). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, et al., 2000) which can be obtained from the World Wide Web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.
Alternatively, the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps (Huang, 1994).
A preferred method for calculating polynucleotide and polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin, et al., 1998).
In one embodiment the genome of the untransformed (wild type) host cell does not prior to transformation with a construct of the invention comprise the one or more LOL genes. In another embodiment the genome of the untransformed (wild type) host cell does not prior to transformation with a construct of the invention comprise a gene homologous to the one or more LOL genes. In another embodiment the untransformed (wild type) host cell or does not prior to transformation with a construct of the invention express the one or more LOL genes.
In one embodiment the host cell, expression construct or polynucleotide comprises and/or expresses at least the
In one embodiment the one or more LOL genes are derived from Epichloë uncinata, Epichloë festucae, Epichloë coenophiala, Epichloë amarillans, Epichloë glyceriae, Epichloë canadensis, Epichloë brachyelytri, Epichloë aotearoae, Epichloë siegelli, Aktinsonella hypoxylon, or Penicillium expansum.
In one embodiment the expression construct, genome or polynucleotide comprises, or the host cell expresses a gene encoding a heterologous acetyltransferase.
In another embodiment the genome of the untransformed (wild type) host cell comprises an endogenous acetyltransferase. In another embodiment the host cell expresses an endogenous acetyltransferase.
Promoters
In one embodiment one or more of the LOL genes are operably linked to a constitutive promoter. In various embodiments the promoter is the histone H3 promoter, the GAPDH promoter, the pna2/tpi hybrid promoter (Aspergillus nidulans or Aspergillus niger), the gpdA promoter (Metarhizium, Aspergillus, or Serendipita), the mbfA promoter, the trpC promoter (Aspergillus nidulans), the hexokinase-1 promoter (Metarhizium robertsii), the class I hydrophobin promoter (Beauveria bassiana) or any other constitutive promoter described herein.
In one embodiment one or more of the LOL genes are operably linked to an inducible promoter. In various embodiments the promoter is the alcA promoter, the alcR promoter, amyB promoter, the gas promoter, the glaA promoter, the niiA promoter, the cbhI promoter, the ctr4 promoter, the thiA promoter or any other inducible promoter described herein.
Those skilled in the art will understand that the different LOL genes may be operably linked to and/or expressed under the control of different promoters and/or terminators.
Loline Alkaloids
In one embodiment the loline alkaloid is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL) and a combination of any two or more thereof.
In one embodiment the loline alkaloid or precursor thereof is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL), (3-amino-3-carboxypropyl)proline (ACPP), exo-1-aminopyrrolizidine (1-AP), exo-1-acetamido-pyrrolizidine (AcAP), and a combination of any two or more thereof.
In one embodiment the loline alkaloid is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-methylloline (NML), N-formylloline (NFL) and a combination of any two or more thereof.
In one embodiment the loline alkaloid or precursor thereof is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-methylloline (NML), N-formylloline (NFL), (3-amino-3-carboxypropyl)proline (ACPP), exo-1-aminopyrrolizidine (1-AP), exo-1-acetamido-pyrrolizidine (AcAP), and a combination of any two or more thereof.
Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.
It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).
This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention. Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.
The invention will now be described by way of example only and with reference to the drawings in which:
The present invention is in part directed to host cells transformed with, having a genome comprising, or transformed with an expression construct comprising, one or more heterologous LOL genes, and methods of producing such host cells. The invention is also directed to recombinant methods for producing one or more loline alkaloids or precursors thereof by culturing a host cell described herein, and methods for producing, or conferring the ability to produce, one or more loline alkaloids to a host cell or organism. The lolines and precursors there of produced by the host cell, and methods of the invention are useful for controlling pests.
The present inventors have for the first time demonstrated production of lolines from heterologous expression of genes in the lolines biosynthetic pathway. This is the first pyrrolizidine alkaloid to be produced in any heterologous host.
As far as the inventors are aware, although there are numerous publications on lolines and loline genes, there is no publication reporting or even considering the production of lolines in a heterologous host.
While there have been some reports of heterologous expression of individual LOL genes, these studies relate to assessing gene function rather than any attempt to produce lolines.
Loline genes have only been reported in Epichloë, Atkinsonella hypoxylon and Penicillium expansum. In Penicillium and Atkinsonella the products of the LOL gene cluster are only predicted, and the cluster is missing some Epichloë LOL gene equivalents. Thus, there is no evidence to suggest that production of the lolines by Epichloë outside of Epichloë itself is possible.
Furthermore, ACPP, the product of the LolC enzyme, is reported to be toxic even to the producer fungus, even w % ben applied at relatively low amounts of 4 mM (Faulkner, et al., 2006). Heterologous expression of lolC (performed to attempt to complement a cystathionine synthase mutant) in Aspergillus nidulans was lost after a single subculture (Spiering, et al., 2005). In addition, attempts to express lolC in E. coli were unsuccessful (Schardl, et al., 2007) and the authors state that these results “suggest that lolC or its enzyme product is toxic to cells”.
The toxicity of lolC or its enzyme product, thus makes the applicants successful production of lolines via the expression of LOL genes including lolC all the more surprising. The applicant's invention therefore additionally provides for pre-selection of strains tolerant to ACPP for use in the heterologous production of lolines.
In addition, the applicants have surprisingly shown that none of the reported LOL genes, perform the step of converting 1-AP to AcAP in heterologous hosts in their experiments. However, the applicants have surprisingly shown that this step can be performed by endogenous enzymatic activity present in some strains. The applicant's invention therefore additionally provides pre-selection of strains capable of performing the conversion of 1-AP to AcAP for use in the heterologous production of lolines.
The term “and/or” can mean “and” or “or”.
The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.
The term “polynucleotide(s),” as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, genes, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. The term also incudes fragments of polypeptides.
A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides.
The term “polypeptide”, as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention, or used in the methods of the invention, may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term also incudes fragments of polypeptides.
A “fragment” of a polypeptide is a subsequence of the polypeptide that in some embodiments performs a function/activity of and/or influences three-dimensional structure of the polypeptide.
As used herein the term “gene” refers to a polynucleotide sequence or its complement that is involved in producing a polypeptide, including regions preceding (leader) and following (trailer) the coding sequence, and introns between individual coding sequence (exons). It also includes to a codon-optimised polynucleotide sequence of the native gene.
The term “constitutive promoter”, as used herein, refers to a promoter that is not regulated and is active in all conditions in the host cell resulting in continuous transcription of its associated gene.
The term “inducible promoter”, as used herein, refers to a promoter that is regulated and is active in the host cell only in response to specific stimuli resulting in transcription of its associated gene.
A “LOL gene” refers to any of the genes that encode an enzyme involved in catalysing a reaction in the loline biosynthetic pathway, as summarised in
In various embodiment the term “LOL gene” encompasses a polynucleotide selected from the group consisting of:
In one embodiment a lolC gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61.
Preferably the lolC gene or variant thereof has at least one of the activity of a gamma-class PLP enzyme encodes and an activity substantially equivalent to that of a lolC gene product.
In a further embodiment the lolD gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62.
Preferably the polypeptide or variant thereof has at least one of the activity of an alpha-class PLP enzyme, and an activity substantially equivalent to that of the lolD gene product,
In a further embodiment the lolF gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63.
Preferably the polypeptide or variant thereof has at least one of monooxygenase activity, and activity substantially equivalent to that of the lolF gene product,
In a further embodiment the lolA gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19.
Preferably the polypeptide or variant thereof has at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product.
In a further embodiment the lolT gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64.
Preferably the polypeptide or variant thereof has at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,
In a further embodiment the lolE gene comprises a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65.
Preferably the polypeptide or variant thereof has at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product.
In a further embodiment the lolO gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66.
Preferably the polypeptide or variant thereof has at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the 1610 gene product.
In a further embodiment the Jo/U gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27.
Preferably the polypeptide or variant thereof has activity substantially equivalent to that of the lolU gene product.
In a further embodiment the lolM gene comprises a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28.
Preferably the polypeptide or variant thereof has at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,
In a further embodiment the lolN gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67.
Preferably the polypeptide or variant thereof has at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product.
In a further embodiment the lolP gene comprises a polynucleotide polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30.
Preferably the polypeptide or variant thereof has at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.
A “LOL gene product” refers to the polypeptide product of any of the genes, i.e. the encoded enzyme, involved in catalysing a reaction in the loline biosynthetic pathway, as summarised in
The term “heterologous” as used herein with reference to a gene or a polynucleotide or polypeptide sequence transformed into or expressed by a host cell or fungus generally means a gene, or a polynucleotide or polypeptide sequence that is not encoded or expressed naturally by the wild type or native host cell or fungus.
The term “heterologous” as used herein with reference to polynucleotides, promoters and terminators, means that such heterologous sequences are not found operably linked to one another in wild type cells in nature. Thus, for example if a promoter is heterologous to the polynucleotide the promoter and polynucleotide are not found operably linked to one another in wild type cells in nature.
The term “host cell” as used herein refers to a fungal cell line cultured as a unicellular entity, which can be, or has been, used as a recipient for LOL genes, and/or expression constructs bearing one or more LOL genes, and/or which can be, or has been, transformed with or subjected to homologous recombination to integrate one or more heterologous LOL genes into the host cell genome. The term includes the progeny of the original host cell which has been transformed or subjected to homologous recombination. It will be appreciated that the progeny of a parent host cell may not be entirely identical in morphology or in genomic or total DNA complement to the original parent.
The term “plant” as used herein encompasses not only whole plants, but extends to plant parts, cuttings as well as plant products including roots, leaves, flowers, seeds, stems, callus tissue, nuts and fruit, bulbs, tubers, corms, grains, cuttings, root stock, or scions, and includes any plant material whether pre-planting, during growth, and at or post-harvest. Plants that may benefit from the application of the present invention cover a broad range of agricultural and horticultural crops, including crops produced using organic production systems.
The term “plant” includes those from any plant species. Such species include gymnosperm species, angiosperm species, and plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants. Such species include those that are used as fodder or forage crops, ornamental plants, food crops, row crops, horticultural crops, fruit crops, vegetable crops, biofuel crops, timber crops, and other trees or shrubs. Such species may be selected from the following: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria. Ananas comosus. Annona spp., Apium graveolens. Arachis spp. Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida). Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape, kale]). Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus. Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia unmflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max). Gossypium hirsutum, Hehanthus spp. (e.g. Helianthus annuus). Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgalum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Pelroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phlewn pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica grananum, Pyrus communis, Quercus spp., Raphanus sativus, Rhewn rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., SaNx sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum betaceum, Solanum integrifoitum or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Trnticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, and Ziziphus spp., among others.
The term “loline alkaloid precursor” as used herein refers to compounds produced as intermediates in the loline biosynthetic pathway. Loline alkaloid precursors may comprise the product of reactions catalysed by the enzymatic product of expression of one or more LOL genes.
The term modified, modify, and grammatical variations thereof, with respect to modifying host cells, or fungi to comprise a polynucleotide, include editing the endogenous genome of the host cells or fungi.
Methods for modifying endogenous genomic DNA sequences are known to those skilled in the art. Such methods may involve the use of sequence-specific nucleases that generate targeted double-stranded DNA breaks in genes of interest.
Examples of such methods include: zinc finger nucleases (Curtin, et al., 2011, Sander, et al., 2011), transcription activator-like effector nucleases or “TALENs” (Cermak, et al., 2011, Mahfouz, et al., 2011, Li, et al., 2012), and LAGLIDADG homing endonucleases, also termed “meganucleases” (Tzfira, et al., 2012).
Targeted genome editing using engineered nucleases such as clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, is an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells (Sander and Joung, 2014). The technique is applicable to fungi (Nodvig, et al., 2015).
The term “substantially equivalent” with reference to any given gene product, enzyme, protein, or polypeptide having activity substantially equivalent to that of any given LOL gene product, preferably means that the gene product, enzyme, protein, or polypeptide is capable of fulfilling the role of the LOL gene product as summarised in Table 1 below, and/or has the enzymatic activity listed in Table 1 below.
The term “control”. “controlling”, “biocontrol” or “biological control” are used interchangeably herein to refer to reduction in growth, growth rate, development, feeding rate, reproduction or number of pests, particularly plant pests, and/or reducing the severity of, or eliminating, symptoms of such pests, particularly symptoms in plants caused by such pests.
The term “(s)” following a noun contemplates the singular or plural form, or both.
Loline alkaloids are produced symbiotically during infection of grasses by endophytes, particularly Epichloë endophytes (which, following a nomenclature realignment, now includes the previously separate anamorph Neotyphodium spp.). These endophytes are considered to be bioprotective, conferring pest, and possibly drought and disease protection to the symbionts of which they form part. Lolines are potent insecticidal compounds and contribute a substantial amount of the bioprotective benefit conferred by Epichloë species that produce them.
Loline alkaloids are 1-aminopyrrolizidines having an oxygen bridge between C2 and C7. The various loline alkaloid variants, namely N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL) are differentiated by substituents on the primary amine.
A loline alkaloid biosynthesis pathway has been proposed as shown in
A loline alkaloid biosynthetic gene cluster has only been identified in fungi belonging to two clades in Pezizomycotina, namely Sordariomycetes (only Epichloë species and Atkinsonella hypoxylon) and Eurotiomycetes (only Penicillium expansum). The Epichloë LOL gene cluster comprises eleven genes, referred to as the LOL genes herein, encoding key enzymes in the loline alkaloid biosynthesis pathway. Homologs to seven of these genes have been reported in P. expansum. The LOL genes are summarised in Table 1 below.
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
festucae
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
E.
uncinata
P.
expansum
P.
expansum
P.
expansum
P.
expansum
P.
expansum
P.
expansum
P.
expansum
The host cells described herein comprise a genome encoding, expressing or having been transformed with one or more LOL genes or expression construct of the invention.
In various embodiments the expression construct of the invention comprises two or more or three or more LOL genes.
In various embodiments the one or more LOL genes are operably linked to one or more regulatory elements that control the transcription, translation or expression of the gene in a host cell transformed with the expression construct. The one or more regulatory elements may be contiguous with the one or more LOL genes or act in trans or at a distance to control the gene of interest.
Suitable regulatory elements include appropriate transcription initiation, termination, promoter and enhancer sequences, or RNA processing signals such as splicing or polyadenylation signals.
Examples of suitable promoters for use in fungal host cells include promoters which are homologous or heterologous to the host cell. Furthermore, suitable promoters for use in the expression constructs of the invention include constitutive promoters, regulatable promoters, inducible promoters or repressible promoters. The promoter may be derived from a gene of the host cell, or a promoter derived from the genes of other fungi, viruses or bacteria. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating expression constructs using genetic constructs comprising the LOL genes of the sequences described herein.
In embodiments where the expression construct comprises two or more LOL genes, or where the host cell comprises or has been transformed with two or more LOL genes, each gene may be under the control of the same promoter or different promoters.
In various embodiments the method comprises transforming the host cell with two or more, three or more, or four or more expression constructs of the invention.
Host cells may be transformed using suitable methods known in the art for achieving heterologous gene expression in fungi and/or yeast. Choice of transformation method will depend on the species and form of the host cell, and the number of expression constructs and or LOL genes to be transformed.
In one embodiment the method comprises transforming the host cell with an expression vector so that the one or more LOL genes is integrated into the genome of the host cell via homologous or non-homologous recombination.
In various embodiments the host cell comprises protoplasts, spheroplasts, spores or conidia.
In one embodiment the method comprises transforming the host cell using polyethylene glycol (PEG)-mediated transformation. Other suitable transformation methods include electroporation, Agrobacterium tumefaciens-mediated transformation, biolistic transformation, or non-PEG-mediated spheroplast transformation.
An exemplary method that may be used to achieve homologous recombination of one or more LOL genes into a fungal host cell genome using sequential transformations is that described by Chiang and co-workers (Chiang, et al., 2013).
Briefly, for genes that are very large and difficult to amplify by PCR, two smaller transforming fragments may be created that fuse by homologous recombination in vivo to reconstruct the full-length coding sequences under the control of a single promoter. Two or more LOL genes may be integrated into the host cell genome using sequential transformations. Each gene or transforming fragment carries a selectable marker to enable selection of transformants that are have been transformed with the gene. Marker recycling may be used so that many genes may be transferred easily into the host cell.
In Vitro
Exemplary methods to produce and at least partially purify and/or isolate one or more of the loline alkaloids or of the invention are described herein. These include the at least partial purification and/or isolation of one or more loline alkaloids from a culture of one or more species, or from culture media or culture supernatants and the like obtained therefrom.
In one embodiment the method comprises maintaining a culture of host cells in the presence of one or more loline alkaloid precursors. For example, in various embodiments the culture is maintained in the presence of one or more of
Choice of culture conditions, including duration of culturing, temperature and/or culture media will depend upon the particular characteristics of the host cell.
The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof.
Background
The Epichloë LOL gene cluster, consisting of 11 genes, has been reported to be required for loline biosynthesis (Spiering, et al., 2005, Pan, et al., 2014). Epichloë lolC was predicted to encode the enzyme that catalyses the first committed step of the loline pathway—the condensation of L-proline with the 3-amino-3-carboxypropyl group from O-acetyl-L-homoserine (Faulkner, et al., 2006). The biosynthetic intermediate produced by this reaction has a dose-dependent effect in E. uncinata—it is toxic to cells when fed at 4 mM, but results in the enrichment of N-formylloline when fed to cultures at 2 mM. P. expansum, the only species outside the Epichloë clade known to carry the loline genes, has homologs to Epichloë lolC,D,F,T,E,O,N. There are no published studies characterizing P. expansum lolC (Pe lolC), but based on its similarity to Epichloë lolC, applicants expect that Pe lolC also encodes an enzyme that catalyzes the same reaction as Epichloë lolC (see Table 2 for amino acid percent identity between E. festucae and P. expansum loline gene products).
The applicants considered that heterologous expression of lolC in fungi which do not possess the LOL gene cluster nor produce lolines, is an important first step towards transforming and expressing selected LOL genes in a non-Epichloë fungus, particularly in light of ACPP's reported toxicity. As initial proof of concept, Epichloë festucae lolC (Ef lolC) was expressed in M. robertsii ARSEF 23 and surprisingly the applicants were able to demonstrate that ACPP was produced. This is described in detail below. The applicants further individually expressed Ef lolC and Pe lolC with successful ACPP production in B. bassiana, A. niger, and T. reesei. S. indica was successfully transformed with Pe lolC, the next step will be confirming expression of lolC. A full summary of expression results of lolC and ACPP production in heterologous hosts is given in Table 5.
Protocol
A plasmid carrying E. festucae lolC gene (SEQ ID NO:31) fused to the constitutively expressed histone H3 promoter (pPH3-lolC) and a plasmid carrying phosphinothricin resistance and green fluorescence (pBAR-GFP) were co-transformed into competent protoplasts of M. robertsii strain ARSEF 23. Transformants carrying pBAR-GFP were selected based on their ability to grow on regeneration medium containing phosphinothricin and green fluorescence. Genomic DNA was extracted from the selected transformants, and presence of pPH3-lolC was confirmed through amplification of Ef lolC in three sets of polymerase chain reactions (PCR).
Two parallel approaches were taken in order to analyse Ef lolC expression and activity in M. robertsii. Firstly, RNA was extracted from four transformed M. robertsii isolates carrying Ef lolC, an isolate that was subjected to the transformation process but does not carry lolC, and the parental strain M. robertsii ARSEF 23, with the aim of observing Ef lolC transcription via RNA extraction, cDNA synthesis, and PCR Secondly, the same strains (above) were grown on M100 medium, freeze-dried, and analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for presence of (3-amino-3-carboxypropyl)proline (ACPP), the suggested product of condensation of L-proline and O-acetyl-L-homoserine (OAH), in the transformant mycelium or medium, indicating the activity of the Ef lolC-encoded enzyme.
Results
Co-Transformation and Identification of Ef lolC Transformants
Thirty (30) transformant isolates that exhibited both phosphinothricin resistance and green fluorescence (a representative set of transformants shown in
Three sets of PCRs, which were done using three different primer combinations on genomic DNA (gDNA) of a subset of the transformants isolated above, identified and confirmed transformants carrying the pPH3-lolC (
Transcription of the Ef lolC Gene
Total RNA extracted from M. robertsii ARSEF 23 and transformant isolates 11, 17, 18, 21, 26 were used for complementary DNA (cDNA) synthesis. In a PCR done using two primers designed to complement two consecutive exons that were separated by an intron, cDNA of transformant isolates 17, 18, 21, 26, produced a 245 bp band, while the pPH3-lolC and gDNA produced a 330 bp band. The size difference between the bands produced by cDNA and gDNA matched the size of the intron, indicating Ef lolC was transcribed to RNA and correctly spliced in transformant isolates 17, 18, 21, and 26. M. robertsii ARSEF 23 and isolate 11, both not carrying Ef lolC as well as controls done with water and without any reverse transcriptase (RT) enzyme during cDNA synthesis did not give any bands (
Chemical Analysis of Ef lolC Transformants
When analysed at m/z 217 (the protonated mass of ACPP), parental strain M. robertsii ARSEF 23 and transformant isolate 11, which did not carry pPH3-lolC, produced two peaks (
Fragmentation analysis of the third peak present in the chromatograms of Ef lolC-expressing isolates matched those with a compound structurally similar to ACPP. This confirmed that the EF lolC transcript was translated to its functional enzymatic form, which catalysed the condensation of proline and OAH to produce ACPP.
The E. festucae lolC gene was heterologously expressed in M. robertsii. This was evidenced by the presence of Ef lolC in transformant gDNA, the presence of the correctly-spliced transcript, and the isolation of ACPP—the suggested end product of the reaction catalysed by the enzyme encoded by Ef lolC. This experimental report marks the first-ever documentation of successful stable heterologous expression of lolC in a non-Epichloë fungus, confirms its role in producing ACPP and demonstrates the potential for producing subsequent steps in the loline biosynthetic pathway, which was in doubt due to the previously suggested toxicity of ACPP.
Introduction
Lolines, which consist of a variable combination of N-acetylnorloline (NANL), N-formylloline (NFL), and N-acetylloline (NAL) in nature, are produced in planta by many endophytic Epichloë species (e.g. E. festucae E2368, E. glyceriae E2772, E. uncinata E167). These alkaloids protect their grass hosts from herbivorous insects, but do not show toxicity towards mammals (Jackson, et al., 1996, Wilkinson, et al., 2000, Schardl, et al., 2013). Blankenship and co-workers (Blankenship, et al., 2001) demonstrated that E. uncinata was able to make lolines in culture, which gave evidence that lolines—and the genes responsible—were of fungal origin. An as yet unidentified plant-encoded acetyl transferase converts loline to NAL, a later ‘decorative’ step that increases the diversity of lolines (Pan, et al., 2014).
In Epichloë, eleven genes (lolC, D, F, A, T, E, O, U, M, N, P) collectively known as the LOL gene cluster, are reported to be required for loline biosynthesis (see
Expression of the eleven genes in the Epichloë LOL cluster was predicted to lead to production of NFL, the end-point of the biosynthetic pathway. Alternatively, eight genes are predicted to be required for production of NANL, which can be converted to an array of lolines. While not predicted to be required for NANL biosynthesis per se, the additional expression of lolA is predicted to increase loline biosynthesis via increasing the biological availability of the precursor molecule OAH.
Outside the Epichloë clade, the loline genes have only been reported in P. expansum (Ballester, et al., 2015, Marcet-Houben and Gabaldon, 2016), wherein the products of the LOL gene cluster have only been predicted. In addition, the P. expansum LOL gene cluster is missing some Epichloë LOL gene equivalents and has homologs of seven of the Epichloë loline genes (lolC, D, F, T, E, O, N).
Production of NANL in a heterologous host in culture vs. production in Epichloë species, is industrially advantageous because of (1) the ability to use a fast-growing heterologous host in a fermenter for continuous mass production of lolines (vs. very little production per day of lolines in vitro and only during the stationary phase by the slow-growing E. uncinata), and (2) the ability to bias production towards individual loline analogues. Lolines have also proven to be very complex to produce via synthetic chemistry (Faulkner, et al., 2006, Cakmak, et al., 2011), thus favouring production via biological means.
The heterologous hosts tested in the reported experiments are (1) E. festucae Fl1, an Epichloë strain that does not possess the loline cluster; (2) B. bassiana strain K4B1, an insect pathogen which is also fermentation-compatible; (3) A. niger strain ATCC 1015, commonly used for industrial fermentation; (4) T. reesei strain RUT-C30 (ATCC 56765), commonly used for industrial fermentation; (5)M. robertsii ARSEF 23, an insect pathogen; (6) Neurospora crassa, a model fungus commonly used for genetic research; (7) S. cerevisiae, a model fungus commonly used for genetic research and industrial fermentation; (8) S. indica, a plant protective endophyte with a broad Angiosperm host range; and (9) U. isabellina, an endophyte. Fungi listed (1)-(7) belong to phylum Ascomycota, while (8) is a member of the phylum Basidiomycota and (9) is a Mucoromycota.
Fungal Strains
Strains used in this study and method of genetic transformation are listed in Table 4. All strains are stored at the Biotelliga laboratory at the University of Auckland, Auckland. New Zealand.
A. niger
B. bassiana
E. festucae
M. robertsii
N. crassa
S. cerevisiae
S. indica
Aspergillus complete
Aspergillus minimal
T. reesei
U. isabellina
Genetic Constructs
A detailed list of transformation constructs is given in Table 11. In brief, all transformed Epichloë loline genes (SEQ ID NO:31 to 33 and 35 to 41) were cloned from E. festucae E2368, except for pBTL10 that, in addition to E. festucae lolD and lolF, contains E. uncinata lolA) coding sequence (from ‘wild type lolA1’—SEQ ID NO 48), pBTL11 that, in addition to E. festucae lolC, lolD and lolF, contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48), pBTL15 that, in addition to E. festucae lolC, contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48), and pBTL57 that contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48). Modified open reading frames [i.e. exons only, codon optimized for Neurospora crassa (using the Codon Optimization Tool at the Integrated DNA Technologies website https://sg.idtdna.com/CodonOpt), and with an HA tag] were also used in some cases. All transformed Penicillium loline genes (SEQ ID NO:68 to 71 and 73) were cloned from P. expansum ICMP 8595. Loline genes were transformed either coupled with a constitutive promoter or with a constitutive promoter and terminator. In a few cases, the gene encoding the selectable marker was present in the same plasmid as the loline genes. In most, however, the appropriate selectable marker was co-transformed with the loline gene constructs. All transformants were selected in media with appropriate selection. PCR screening was done to test for the presence of the loline genes on gDNA preparations done according to standard DNA extraction (miniprep gDNA extraction).
RNA Extraction and qPCR
RNA was extracted from fungal mycelia using the TRIzol® reagent (Life Technologies) according to the manufacturer's protocol. RNA was either DNased with DNase I recombinant (Roche) and used for cDNA synthesis with iScript™ (Biorad) or was DNased and cDNA synthesised using the iScript™ gDNA Clear cDNA Synthesis kit (Biorad). qPCR was done as per standard Biotelliga laboratory protocol using SsoAdvanced™ Universal SYBR Green Supermix (Biorad).
Chemical Analysis
Production of relevant compounds (ACPP, 1-AP, AcAP, and lolines) by transformed fungi were detected with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The values of detected compounds are represented in micromolar (μM) and were calculated using the following formula:
Concentration in μM=(Concentration in μg/ml÷molecular weight)×1000
The molecular weights of the compounds are: 216 (ACPP), 126 (1-AP), 168 (AcAP), 182 (NANL), 154 (loline), and 182 (NFL).
Results
Expression of lolC Results in Production of ACPP in Heterologous Hosts
Of the non-Epichloë fungi tested, A. niger. B. bassiana. M. robertsii, and T. reesei produced ACPP constitutively in culture upon expression of Ef lolC (Table 5). A. niger. B. bassiana, and T. reesei also produced ACPP constitutively in culture upon expression of Pe lolC (Table 5). Pe lolC was not attempted to be transformed into M. robertsii. The ACPP of biological origin was identical to chemically synthesised ACPP. It was observed in typical extracted chromatograms for the protonated mass of ACPP (m/z 217) in transformants with lolC, compared to wild type and a transformant not carrying lolC, but with the same selectable marker (
A. niger
A. niger
B. bassiana
B. bassiana
M. robertsii
N. crassa
N. crassa
S. cerevisiae
S. cerevisiae
S. cerevisiae
T. reesei
T. reesei
Expression of Epichloë Loline Pathway Genes Results in Loline Production in Heterologous Hosts
Based on their ability to produce ACPP upon expression of lolC, heterologous hosts A. niger, B. bassiana, and M. robertsii, along with E. festucae Fl1, were selected as candidate heterologous hosts for expression of lolCDFAITEOU—or subsets of genes thereof. The lol genes transformed were obtained from E. festucae E2368, E. uncinata AR 1006 and/or P. expansum ICMP 8595. The industrial A. niger strain ATCC 1015, was transformed with Epichloë lolD. F,T,A,I,O. Cultures were supplemented with 1 mM ACPP, and upon feeding, successfully produced 0.385 μM NANL (Table 6).
Transformation of Epichloë lolCDFA1TEU to B. bassiana resulted in production of 0.179 μM AcAP when fed with 2 mM ACPP (see
A. niger
B.
bassiana
B.
bassiana
B.
bassiana
E.
festucae
Expression of P. expansum Lol Genes Result in Loline Production in Heterologous Hosts
Heterologous hosts A. niger and B. bassiana, which were capable of producing ACPP by expression of Pe lolC (Table 5), were transformed with Epichloë to lolDFTA1, or P. expansum to lolDFTO, henceforth Pe to lolDFTO, or subsets of these genes thereof. All genes in transformants were confirmed to be expressed using qRT-PCR. Both A. niger and B. bassiana successfully produced loline pathway intermediates ACPP, I-AP, and AcAP in the transformant strains expressing the Pe lolCDFTO genes (see Table 7 for details). However, in both A. niger and B. bassiana, transformants did not produce any detectable levels of NANL despite producing relatively high amounts of the precursor intermediate AcAP. This was unexpected as these transformants were expressing Pe lolO≥1-fold relative to actin, compared to previous transformants which produced detectable levels of NANL with even ≤1-fold expression of Ef lolO (relative to actin). This may indicate that, at least in heterologous systems. Pe lolO is less efficient than Ef lolO in converting AcAP to NANL. Therefore, Ef lolO was transformed to B. bassiana transformants already expressing Pe lolCDFTO gene array. Biological triplicates of 17 transformants that resulted from transformation of Ef lolO to the Pe CDFTO background were screened for Ef lolO and Pe lolO expression levels. From these, 12 transformants with the highest Ef lolO/Pe lolO expression were selected for testing gene expression of the remaining loline genes. (see Table 8 for expression of Pe lolC, D, F, T, O and Ef lolO in biological triplicates of the 12 transformants). Variable gene expression further supported selection against toxic intermediate production in this system and highlighted the requirement for careful selection of transformants with appropriate gene expression ratios. A transformant (no. 17 in Table 8), which did not express Pe lolC, but expressed all five subsequent genes, was fed with 2 mM ACPP and produced 3.63 μM NANL.
A. niger
A. niger
B. bassiana
B. bassiana
A native N-acetyltransferase of the heterologous host converts 1-AP to AcAP
Epichloë lolCDFTEOUA1 were transformed to heterologous hosts in accordance with the general scientific consensus that the former seven genes from Epichloë are necessary and sufficient to produce NANL, and that lolA, while not necessary for NANL production, would increase precursor OAH levels resulting in a good flux of intermediates through the pathway. However, existing experimental evidence does not fully clarify the function and necessity of lolD, lolF, lolU, and lolE for NANL production (see Table 9 for summary and references to experimental evidence of loline gene functions).
The roles of lolF and lolD are well supported by biochemical theory that the pyrrolodinium ions, which are the putative products of LolD- and LolF-catalysed reactions, are likely to be highly unstable compounds not able to be synthesised for authentic standards or detected by targeted LC-MS/MS (D. Rennison, personal communication, 2016). Therefore, the applicants focused on clarifying the function of lolE and lolU.
lolU was considered by the applicants to be a possible candidate for the acetylation of 1-AP to AcAP, based on the identification of an HMM signature of CoA-dependent acyltransferases superfamily in InterPro (https://www.ebi.ac.uk/interpro/) and the top hit to acetyltransferase in Swiss-MODEL structural analysis (https://swissmodel.expasy.org/). When fed with 2 mM 1-AP, AcAP was detected in two independent B. bassiana transformants which were expressing E. festucae E2368 lolU, as well as in two independent transformants without lolU. No AcAP was observed in the growth medium with 2 mM 1-AP control. No statistical difference in 1-AP acetylation was observed between lolU-containing and non-containing B. bassiana K4B1. To further explore this preliminary data that showed that an N-acetyltransferase gene native to the host is capable of the acetylation of 1-AP to AcAP, a 1-AP feeding assay was done using a range of fungi. No ACPP or hydroxy-AcAP was detected in any culture. However, wild type strains of all fungi tested except for E. festucae Fl1, S. cerevisiae and S. indica, were capable of acetylating 1-AP to AcAP (see Table 10 for details). All other Epichloë strains tested (E. uncinata AR1006 and E. festucae E2368), and an Fl1 strain carrying Ef lolDFTEOU were capable of the acetylation however, which may indicate that the Fl1 strain tested may have converted 1-AP to AcAP, albeit less efficiently—and thus at levels less than the detection threshold. While the ability of wild type fungi to convert 1-AP to AcAP without lolU is not conclusive evidence that lolU plays a role in acetylation of 1-AP to AcAP in the native system, it is proof that a native acetyltransferase, perhaps universally present across Kingdom Fungi, has the ability to convert 1-AP to AcAP.
A. niger
A. niger
B. bassiana
B. bassiana
B. bassiana
B. bassiana
B. bassiana
B. bassiana
E. festucae
E. festucae
E. festucae
E. festucae
E. festucae
E. festucae
E. uncinata
E. uncinata
E. nigrum
E. nigrum
P. expansum
P. expansum
R. solani
R. solani
Rhizopus sp.
Rhizopus sp.
S. zeae
S. zeae
T. reesei
T. reesei
S. indica
S. indica
U. isabellina
U. isabellina
K. marxianus
K. marxianus
S. cerevisiae
S. cerevisiae
Epichloë lolE, the only other nonheme iron dioxygenase besides lolO present in the Clavicipitaceae loline cluster, is suggested to be ‘not absolutely required’ in the ether bridge formation due to unpublished observations with a lolE knockout mutant (alluded to in (Pan, et al., 2014)). However, recently it was reported that LolE has no role in vitro in the two oxygenation steps required to form NANL from AcAP (Pan, et al., 2018). But, there is no published data to provide conclusive evidence to whether LolE plays a role in vivo in one of the two oxygenation steps that are required to form NANL from AcAP. To this end, B. bassiana transformants expressing either lolE, lolO, or lolE and lolO together (‘lolEO’) were obtained, fed with 0.8 mM 1-AP, and the mycelial fraction was analysed for compounds of interest. All transformants as well as wild type B. bassiana fed with 1-AP produced AcAP. However, the intermediate generated by hydroxylation of AcAP (hydroxy-AcAP) and NANL were only detected in lolO and lolEO transformant cultures. No AcAP, hydroxy-AcAP, or NANL was detected in the growth medium with 0.8 mM 1-AP control. There was no correlation of lolE expression with hydroxy-AcAP levels in lolEO cultures (R2=0.0009).
Discussion
The study of the current Example sought to achieve three main goals: (1) produce ACPP in heterologous fungal hosts by expression of the lolC gene; (2) produce NANL in heterologous hosts selected due to their ability to form ACPP; and (3) understand the requirement for lolE and Jo/U in the Clavicipitaceae loline production pathway.
When attempting to produce ACPP by expression of Epichloë lolC, the applicants observed that the native E. festucae gene with the introns and exons expressed under a constitutive promoter consistently resulted in a detectable lolC transcript in all filamentous fungi tested. Transcription of the gene led to ACPP production in culture in B. bassiana, M. robertsii, and the industrial strains A. niger and T. reesei (F. festucae Fl1 was not tested with the E. festucae lolC cassette). Although transcripts were detected in N. crassa, no ACPP was detected. The modified lolC—i.e., Ef lolC exons only, codon-optimized for N. crassa—resulted in variable levels of transcription in the tested hosts and was unsuccessful in producing ACPP in all cases. The highest amount of ACPP observed in a heterologous host expressing only Ef lolC was 1390 μM, produced by a B. bassiana transformant. Expression of Pe lolC was attempted only in A. niger. B. bassiana, and T. reesei, and resulted in successful transcription of Pe lolC and ACPP production in all three cases. The highest amount of ACPP observed in a heterologous host expressing Pe Jo/C only was 1844 μM, produced by a B. bassiana transformant. The maximum amount of ACPP observed in any transformant to-date is 2463 μM, which was produced by a B. bassiana transformant carrying Pe lolCDFTO and Ef lolO. It is noteworthy that endogenous ACPP amounts ≥3 mM has not been observed in any heterologous host tested so far. This may be due to the dose-dependent cytotoxic effect of ACPP, which has been reported for E. uncinata in previous literature (Faulkner, et al., 2006).
The applicants successfully produced NANL-loline-NFL, and NANL by itself, as well as the full array of chemically detectable pathway intermediates in B. bassiana via expression of Epichloë lolCDFAITEOUMNP and Epichloë lolCDFAITEOU, respectively. To the best of the applicant's knowledge, this is the first report of heterologous production of the full loline pathway in a non-native host. NANL was also produced in transformants expressing Epichloë lolDFTA1O in the industrial A. niger strain ATCC 1015, those expressing Ef lolDFTEOU in E. festucae strain Fl1, and those expressing the Pe lolDFTO-Ef lolO combination in B. bassiana strain K4B1. The latter three cultures were all fed 1 or 2 mM ACPP as they all lacked lolC. All detectable loline pathway intermediates up to NANL, were produced by A. niger and B. bassiana transformants that were expressing Pe lolCDFTO as well, although detectable levels of NANL was absent. The Epichloë loline genes transformed to the heterologous hosts originated from E. uncinata AR1006 (Eu lolA1) and/or E. festucae E2368 (Ef lolCDFTEOUMNP), which has currently been observed to produce lolines in planta only. All Penicillium lol genes are from P. expansum, the only Penicillium species in which the lol genes have been reported to-date. To the best of the applicant's knowledge, the current study is also the first report of successful heterologous expression of any Pe lol gene.
Analysis of loline gene expression data showed possible bottlenecks in expression of genes such as lolF and lolO. Re-transformation of lolO under the E. festucae Fl1 histone H3 promoter that was previously seen to produce relatively ‘high’ gene expression, increased the NANL level from 0.385 μM in Bb H15 to 1.209 μM in Bb O16. A further increase in production in the BbO16 strain was achieved by feeding with iron (in the form of ammoniumiron(II)sulfate hexahydrate) and 2-oxoglutarate, which have been shown to bias the oxygenation reaction catalyzed by lolO-encoded enzyme towards NANL (Pan, et al., 2018).
The applicants saw that both wild type B. bassiana without any loline genes and B. bassiana lolU transformants accumulated AcAP when fed with 1-AP. This is consistent with the previous observation that there was no obvious change in the loline alkaloid profile of lolU RNAi transformants (Pan, 2014). Therefore, the applicants conclude that lolU, although a putative acetyl transferase, is not exclusively responsible for the acetylation of 1-AP to AcAP, and that a native gene of the heterologous host is capable of this conversion. The applicants also observed no lolU expression in transformant Bb O16, although it produced NANL. Collectively, these observations make the necessity of lolU in the loline pathway questionable.
Production of NANL in A. niger ATCC 1015 strain is of significance as it is a strain well-suited—and commonly used—in fermentation industry. B. bassiana, the heterologous host which has produced the highest levels of lolines in the systems tested so far, is reported as a generalist systemic endophyte. Suitability when using as a spray treatment, lifespan in soil vs. foliage, effect on plant growth, and ability to form a loline-producing but non-sporulating mutant all remain basic questions to be answered.
Based on our observations, a two-step screen, which consists of checking the ability of a selected fungus to (1) produce ACPP through expression of lolC and (2) convert 1-AP to AcAP without any transgenes, is proposed when selecting a heterologous host for expression of lolines. If a fungus is capable of both, and is fermentation compatible and/or is an established endophyte, it may be an optimal heterologous host for loline production. The best experimental approach should be then determined based on the ACPP- and 1-AP-feeding experiment results and availability of selectable markers for the selected fungus. If a fungus with a high potential as an endophyte or fermentation compatibility is capable of producing ACPP, but is unable to convert 1-AP to AcAP, it may be useful to check if it could accumulate ‘high’ levels of 1-AP. If so, it may be used in a sequential fermentation, where the 1-AP formed is converted to the next products of the pathway by a co-cultivated host. Alternatively, if a gene that can convert 1-AP to AcAP is identified, it can be expressed in the fungus along with the other loline genes, thus giving it the ability to make the desired end product. On the other hand, if a fungus is capable of the 1-AP to AcAP conversion, but cannot produce ACPP, it may be useful to determine if ACPP-toxicity has led to the mortality of ACPP-producers and if so, an approach which transforms other desired loline genes prior to transformation of lolC, and transformation of lolC last could be considered. A case-by-case review of risks vs. benefits for each scenario is recommended.
Following the experiments of this example, the applicants postulate that, contrary to previous expectation for requirement of seven genes, expression of just five genes—lolC, lolD, lolF, lolT, lolO—in a heterologous host should result in the production of NANL—the first fully cyclized loline intermediate of the pathway.
Aureobasidium
pullulans
M. robertsii
E. festucae
N. crassa, CDS, C
M. robertsii
E. festucae
N. crassa, CDS, C
M. robertsii
E. festucae
N. crassa, CDS, C
M. robertsii
E. festucae
N. crassa, CDS, C
M. robertsii
E. festucae
N. crassa, CDS, C
M. robertsii
E. festucae
N. crassa, CDS, C
E. festucae Fl1
E. festucae
S. cerevisiae
E. festucae
N. crassa, CDS
E. festucae
E. festucae
E. festucae
E. festucae
E. festucae
E. festucae
N. crassa, CDS
E. festucae
N. crassa, CDS
E. festucae
N. crassa, CDS
E. festucae
E. festucae Fl1
E. festucae
E. festucae Fl1
E. uncinata
M. robertsii
E. festucae
M. robertsii
E. festucae
E. festucae Fl1
E. festucae
E. festucae Fl1
E. uncinata
M. robertsii
E. festucae
M. robertsii
E. festucae
A. nidulans
E. festucae
M. robertsii
E. festucae
S. cerevisiae
E. festucae
s.
cerevisiae, CDS, C
E. festucae
N. crassa, CDS
M. robertsii
E. uncinata
E. festucae Fl1
E. festucae
E. festucae Fl1
E. festucae
B. bassiana
E. festucae
M. robertsii
E. festucae
E. festucae Fl1
E. festucae
B. bassiana
E. festucae
M. robertsii
E. festucae
E. festucae Fl1
E. festucae
B. bassiana
E. festucae
E. festucae
E. festucae Fl1
E. festucae
E. festucae Fl1
E. festucae
E.
festucae Fl1
E. festucae
M. robertsii
E. festucae
B. bassiana
E. festucae
B. bassiana
E. festucae
M. robertsii
E. uncinata
E. festucae Fl1
E. festucae
E. festucae Fl1
E. festucae
B. bassiana
E. festucae
M.
robertsii
E. festucae
E. festucae Fl1
E. festucae
B. bassiana
E. festucae
M. robertsii
E. festucae
E. festucae Fl1
E. festucae
E.
festucae Fl1
P. expansum
E. festucae Fl1
P. expansum
E. festucae Fl1
P. expansum
E. festucae Fl1
P. expansum
E. festucae Fl1
P. expansum
S. indica
P. expansum
M. robertsii
E. festucae
M. robertsii
E. festucae
A. nidulans
E. festucae
M. robertsii
E. festucae
Introduction/Summary
The protein encoded by lolU was considered a possible candidate for the acetylation of 1-AP to AcAP, based on its structural similarity to N-acetyltransferases. However, when fed with 2 mM 1-AP, AcAP was detected in two independent B. bassiana transformants which were expressing E. festucae E2368 lolU, as well as in two independent transformants without lolU No AcAP was observed in the growth medium with 2 mM 1-AP control. This indicated that an N-acetyltransferase gene native to the host was capable of the acetylation of 1-AP to AcAP. As this gene is yet unidentified, the ability to convert 1-AP to AcAP is currently a required characteristic for a given fungus to successfully produce NANL. Therefore, a screen of multiple fungal taxa for the ability to convert 1-AP to AcAP was proposed. This screen has been done with a set of fungi from phylum Ascomycota, to which the native loline producer Epichloë species belong, and a fungus from unplaced subphylum Mucoromycota (formerly Zygomycota). All strains tested, except Epichloë festucae strain Fl1 were able to acetylate 1-AP to form AcAP.
Fungal Strains
All fungal strains (Table 12) were sub-cultured onto potato dextrose agar (PDA) from original or glycerol stocks where possible. All subsequent sub-cultures were also performed with PDA.
Aspergillus niger
Beauveria
bassiana
Beauveria
bassiana
Beauveria
bassiana
Epichlo{umlaut over (e)}
festucae
Epicoccum sp.
Metarhizium
robertsii
Penicillium
expansum
Rhizoctonia
solani
Rhizopus sp.
Sarocladium spp.
Trichoderma
reesei
Establishing Optimal Media for all Fungal Strains
A preliminary growth study for six wild type strains of fungi was performed using two complex media, PD broth (PDB) and sabouraud dextrose broth (SDB). The strains used were A. niger, B. bassiana, E. festucae, M. robertsii, P. expansum, and T. reesei.
A small square of fungi (approximately 0.5 cm2) was added to 1 ml of MilliQ water in a sterile bead beating tube. The mycelial suspension was macerated in a tissue homogenizer for 30 seconds at 4,000 RPM. 40 μl of this mycelium was then added to four 50 ml Falcon tubes containing 2 ml of SDB or PDB in duplicate.
After one day the cultures were observed and growth was recorded. Those with significant biomass were fed with 22 μl of 60% w/v ethanol. Three days post-inoculation, the cultures were again observed and growth noted. Overall growth was measured using a subjective scale.
Fungal Cultures and Inoculation
All fungi, excluding numbers 5 and 7, were inoculated in batches, based on qualitative observations of their growth. There are three groups, with fast, medium, and slow growing species. Each fungus was correspondingly inoculated so that the feeding of 1-AP could all be performed at the same time for all species. A small square of fungi approximately 1 cm2) was added to 1 ml of MilliQ water in a sterile bead beating tube. This tube was then bead beaten in a tissue homogenizer for 30 seconds at 4,000 RPM. 40 μl of this mycelium was then added to five 50 ml Falcon tubes containing 2 ml of SDB; duplicate treatment and triplicate treatment cultures. The lids were loosely fitted on the tubes and secured with sellotape. These tubes were then incubated in an upright rack at 25° C. with shaking at 125 RPM in the dark. Four media-only controls were also incubated in these conditions.
Feeding of 1-AP
1-AP solution sufficient for the required number of treatments to give a final concentration of 2 mM was run through a vacuum pump for three minutes to concentrate it, leaving a solution containing roughly 60% w/v ethanol. This 1-AP solution was then fed in equal amounts to the treatment cultures, and an equivalent volume of 60% w/v ethanol added to the control tubes. All tubes were incubated in the same conditions for 48-72 h before harvesting.
Harvesting of Chemical Samples
One ml of mycelium and broth was macerated by bead-beating in a Precellys homogenizer, then filtered through a 0.2 μm syringe filter. Filtrates were then analysed for loline intermediates via LC-MS/MS.
Biomass Measurements
For each control tube, the dry biomass was determined; the filter papers were allowed to incubate at room temperature for several days before weighing.
Results
Biomass Measurement
Individual weights of a set of labelled filter papers were recorded. Mycelium was sterilized holding ≥20 min in 10% Prevail®, and was added to the corresponding filter paper and was incubated at 60° C., overnight. As some of these papers had brown charred sections were lighter than expected, they were left on the bench for three more days and were weighed again. The average dry biomass weight for all species ranged between 22 and 52 mg. The highest biomass was produced by A. niger ATCC 1015, and the lowest by E. festucae Fl1 and Rhizopus spp.
Chemistry Analysis
Initially, eight different strains were analysed to test for their endogenous ability to convert 1-AP to AcAP. Of these eight, seven were found to be able to perform this conversion, with the exception being E. festucae F11. Roughly 0.5-3 percent of 1-AP was converted to AcAP. The highest average amount of AcAP measured was 267 μM by for K. marxianus; and the lowest was 9.1 μM by A. niger ATCC 1015 (Table 10).
Chemistry samples for B. bassiana K4B1 lolU #20, R. solani ICMP 17586, and Sarocladium spp. BTL-E218 were harvested, but not analysed.
In the current screen conducted, all fungal species tested, except E. festucae Fl1 strain, were capable of acetylating 1-AP to AcAP. The amount of AcAP produced by different strains was low but still highly variable among species, with the production of AcAP by T. reesei in this study the highest detected from any 1-AP feeding study or produced by heterologous hosts in any instance (
The expression constructs, host cells, and methods of the invention have utility for many agricultural, horticultural, medical and veterinary applications, such as providing horticulturalists with a useful means of controlling plant pests, and providing therapies for the treatment or prevention of insect infection or infestation in humans or non-human animals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 738615 | Dec 2017 | NZ | national |
This application is a continuation of U.S. patent application Ser. No. 16/956,476, filed Jun. 19, 2020, which is a U.S. National Stage Application filed under 35 U.S.C. § 371 of International Application No. PCT/IB2018/060481, filed Dec. 21, 2018, which claims the benefit of New Zealand Application No. 738615, filed Dec. 21, 2017. All of these applications are hereby incorporated by reference in their entireties
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| Number | Date | Country | |
|---|---|---|---|
| 20220356498 A1 | Nov 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16956476 | US | |
| Child | 17879663 | US |
