Patent Application
20210332345
This application claims the benefit of priority of Singapore application No. 10201807514P, filed 31 Aug. 2018, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The invention is in the field of biotechnology. In particular, the invention relates to methods for the discovery of fungal terpene synthases and the use of fungal terpene synthases for the production of terpenoids.
Terpenoids constitute one of the most structurally diverse classes of natural products with wide applications as pharmaceuticals (such as Taxol and artemisinin), as food coloring (such as carotenoids), flavors and fragrances (such as nootkatone and sclareol) and biofuels (such as farnesene). The terpenoid diversity is attributed primarily to terpene synthases (TPSs), which convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic terpene scaffolds. Specifically, the terpene skeletal diversity arises from two main features of TPSs: a large number of TPSs with vastly different functions and the ability of many TPSs to catalyze multiple terpene products from a single substrate. Almost half of the characterized monoterpene and sesquiterpene synthases produce significant amounts of additional products, apart from their main products.
Structurally, all sesquiterpene synthases (STSs) from plants, fungi and bacteria have a conserved metal binding motif (unlike plant STSs are DDXXD, fungal STSs are DE(N)XXD) and NSE triad. However, outside of the motif, there is limited sequence similarity between plant and microbial TPSs. Many plant TPSs have been discovered and studied over the past few decades, however, the study of fungal terpene synthases was lagging behind. Currently, only a handful of fungal TPS have been cloned and functionally characterized (less than 50, Table 1).
Fungi have enormous diversity (-5 million species) and outnumber plants by at least 10 times. Each fungus has an average of 10-20 putative TPS homologs in Basidiomycota, indicating that fungal terpenoids and TPS genes represent rich but largely untapped natural resources. In addition to the discovery of novel TPS genes and terpenoids, it is also valuable to identify other potentially more efficient TPSs than existing TPSs with the same functions or products. This is due to the various applications of terpenoids and huge commercial interests in terpenoids. In some cases, the production of terpenoids was limited by insufficient activity of terpene synthases. Identification of TPS enzymes with novel products, superior activity and selectivity would greatly benefit the industrial biotechnology society for terpenoid production.
In one aspect, there is provided a bacterial strain comprising one or more vectors encoding
a) one or more enzymes to produce one or more terpene precursors; and
b) a fungal terpene synthase (FTPS).
In another aspect, there is provided a genetically modified 1-deoxyxylulose-5-phosphate synthase (DXS) enzyme, wherein the genetic modification is a mutation at one or more amino acid positions.
In another aspect, there is provided a genetically modified fungal terpene synthase (FTPS), wherein the genetic modification is a mutation at one or more amino acid positions.
In another aspect, there is provided a method of producing a terpenoid comprising a) culturing the bacterial strain as described herein in an expression medium, and b) isolating the terpenoid from said expression medium.
In another aspect, there is provided a method of producing a terpenoid comprising a) culturing a bacterial strain comprising a vector encoding the genetically modified FTPS as described herein in an expression medium and b) isolating the terpenoid from said expression medium.
In another aspect, there is provided a fungal terpene synthase (FTPS) encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:39.
In another aspect, there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:39.
As used herein, the term “terpene” refers to a class of organic compounds produced by plants, bacteria, fungi and insects. The building blocks of terpenes have a five-carbon isoprene unit.
As used herein, the term “terpenoid” refers to a large and diverse class of organic compounds derived from terpenes and include terpenes. The building blocks of terpenes have a five-carbon isoprene unit and contain additional functional groups, typically oxygen-containing functional groups. Terpenes are a subset of terpenoids.
As used herein, the term “terpene precursor” refers to a substrate that is converted to a terpene or terpenoid by a terpene synthase. The substrate may be converted to a terpene or a terpenoid.
As used herein, the term “terpene synthase” (TPS) refers to an enzyme that converts terpene precursors to terpenes and/or terpenoids. The term “fungal terpene synthase” (FTPS) refers to a terpene synthase that is isolated from a fungus.
As used herein, the term “UP” in the context of a fungal terpene synthase (FTPS) refers to a domain of the FTPS. The UP domain is situated upstream of the DW domain.
As used herein, the term “DW” in the context of a FTPS refers to a domain of the FTPS. The DW domain is situated downstream of the UP domain.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
In a first aspect, the present invention refers to a bacterial strain comprising one or more vectors encoding a) one or more enzymes to produce one or more terpene precursors, and b) a fungal terpene synthase (FTPS).
It will be appreciated by a person skilled in the art that the one or more vectors comprise polynucleotide sequences that encode the one or more enzymes to produce one or more terpene precursors and FTPS.
The polynucleotides encoding the one or more enzymes to produce one or more terpene precursors and the FTPS may be located on separate vectors, on a single vector or combinations thereof. In one embodiment, the polynucleotides encoding the one or more enzymes to produce one or more terpene precursors are in a single vector and the polynucleotide encoding the FTPS is in a separate vector.
In one embodiment, the one or more vectors comprise one or more nucleotide sequences encoding the one or more enzymes and the FTPS, operably linked to a promoter.
In some embodiments, the promoter is a constitutive promoter or an inducible promoter. In a preferred embodiment, the promoter is an inducible promoter. Examples of inducible promoters include but are not limited to T7 RNA polymerase promoter, araBAD promoter, a lac promoter, a trp promoter and a Tac promoter (ptac) or the variants of these promoters.
In a preferred embodiment, the inducible promoter is T7 RNA polymerase promoter.
In one embodiment, the one or more enzymes to produce the one or more terpene precursors are part of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. It will be appreciated to a person of skill in the art that the DXP pathway is also referred to as the non-mevalonate pathway, the mevalonate-independent pathway or the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway. The DXP pathway converts pyruvate and glyceraldehyde-3-phosphate to terpene precursors and the enzymes in this pathway include DOXP synthase (DXS), DXP reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), HMB-PP synthase (ispG), HMB-PP reductase (IspH) and isopentenyl diphosphate isomerase (IDI).
In a preferred embodiment, the enzyme is 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl diphosphate isomerase (IDI) or both.
In one embodiment, the DXS comprises the amino acid sequence set forth in SEQ ID NO: 6.
In some embodiments, the DXS may be genetically modified. The genetic modification may be a mutation at one or more amino acid positions of the amino acid sequence encoding the DXS. In some examples, the mutation is an amino acid substitution, insertion, deletion or combinations thereof.
In some embodiments, the genetically modified DXS has a higher solubility than an unmodified or wild-type DXS.
In some embodiments, the mutation is selected from the group consisting of H105T, E210D, Q459L, L415T and a combination thereof of SEQ ID NO: 6.
In a preferred embodiment, the mutation is H105T.
In another preferred embodiment, the mutation is E210D, Q459L and L415T.
In one embodiment, the genetically modified DXS comprises the amino acid sequence set forth in SEQ ID NO: 24 or 25.
In one embodiment, the DXS comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 24 or 25.
In one embodiment, the one or more enzymes to produce the one or more terpene precursors is expressed at an elevated level compared to a wild type enzyme. The one or more enzymes may be genetically modified.
In some embodiments, the terpene precursors described herein is farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), or combinations thereof.
In a preferred embodiment, the terpene precursors are FPP and/or GPP.
The bacterial strain described herein comprises one more vectors encoding a fungal terpene synthase (FTPS). In a preferred embodiment, the FTPS is a monoterpene synthase or a sesquiterpene synthase. In a further preferred embodiment, the FTPS is a linalool synthase, a nerolidol synthase or a linalool and nerolidol synthase (LNS).
In some embodiments, the FTPS is isolated from Agrocybe aegerita, Agrocybe pediades, Galerina marginata, Hypholoma sublateritium, Dendrothele bispora, Moniliophthora roreri, Piloderma croceum, Sphaerobolus stellatus, Coprinopsis cinerea, Omphalotus olearius, Fomitopsis pinicola, Stereum hirsutum, Fusarium graminearum, Fusarium fujikuroi, Fusarium sporotrichioides, Aspergillus terreus, Penicillium roqueforti, Hypoxylon sp., Armillaria gallica, Botrytis cinerea, Daldinia eschscholzii or combinations thereof.
In one embodiment, the FTPS is isolated from Agrocybe aegerita, Agrocybe pediades, Galerina marginata, Hypholoma sublateritium, Hebeloma cylindrosporum or combinations thereof.
In a preferred embodiment, the FTPS is isolated from Agrocybe aegerita or Agrocybe pediades.
In some embodiments, the FTPS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.
In some embodiments, the FTPS comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39.
In one example, the bacterial strain described herein contains an FTPS that is expressed at a higher level than a wild-type FTPS.
In some embodiments, the FTPS may be genetically modified. The genetic modification may be an amino acid substitution, insertion, deletion, C-terminal truncation, N-terminal truncation or combinations thereof. The mutation may be one or more mutations in the UP domain of the FTPS, one or more mutations in the DW domain of the FTPS, or both. The UP and DW domains would be understood by the skilled person to vary based on the fungal strain. In one example, the UP domain of the FTPS isolated from Agrped1_689675 (SEQ ID NO: 3) is characterized by amino acid positions 1-170. In another embodiment, the UP domain of the FTPS isolated from Agrped1_689671 (SEQ ID NO: 2) is characterized by amino acid positions 1-169. In yet another embodiment, the DW domain for the FTPS isolated from Agrped1_689675 (SEQ ID NO: 3) is characterized by amino acid positions 171-325. In yet another embodiment, the DW domain of the FTPS isolated from Agrped1_689671 (SEQ ID NO: 2) is characterized by amino acid positions 170-324.
In a preferred embodiment, the mutation is F204D, F204G, F204R, F204I, F204L, F204V or combinations thereof of SEQ ID NO: 3.
In some embodiments, the genetically modified FTPS comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16 SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.
In some embodiments, the FTPS comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.
In some embodiments, the bacterial strain is modified or genetically modified.
In one embodiment, the bacterial strain described herein is Escherichia coli.
In one aspect, the present invention refers to a genetically modified 1-deoxyxylulose-5-phosphate synthase (DXS) enzyme, wherein the genetic modification is a mutation at one or more amino acid positions. In one embodiment, the mutation described herein is an amino acid substitution or insertion or deletion. In yet another embodiment, the mutation is selected from the group consisting of H105T, E210D, Q459L, L415T and a combination thereof of SEQ ID NO: 6.
In a preferred embodiment, the mutation is E210D, Q459L and L415T.
In another preferred embodiment, the mutation is H105T.
In another aspect, the present invention refers to a genetically modified DXS enzyme comprising an amino acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 25.
In yet another aspect, the present invention refers to a genetically modified DXS enzyme comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to an amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25.
In one aspect, the present invention refers to a genetically modified fungal terpene synthase (FTPS), wherein the genetic modification is a mutation at one or more amino acid positions.
The genetically modified FTPS is modified relative to a wild type or unmodified FTPS. In one embodiment, the unmodified FTPS is isolated from Agrocybe aegerita, Agrocybe pediades, Galerina marginata, Hypholoma sublateritium, Hebeloma cylindrosporum or combinations thereof.
In a preferred embodiment, the unmodified FTPS is isolated from Agrocybe aegerita or Agrocybe pediades.
In some embodiments, the mutation described herein is an amino acid substitution, insertion, deletion, C-terminal truncation, N-terminal truncation or combinations thereof. The mutation may be one or more mutations in the UP domain of the FTPS, one or more mutations in the DW domain of the FTPS, or both. The UP and DW domains would be understood by the skilled person to vary based on the fungal strain. In one example, the UP domain of the FTPS isolated from Agrped1_689675 (SEQ ID NO: 3) is characterized by amino acid positions 1-170. In another embodiment, the UP domain of the FTPS isolated from Agrped1_689671 (SEQ ID NO: 2) is characterized by amino acid positions 1-169. In yet another embodiment, the DW domain for the FTPS isolated from Agrped1_689675 (SEQ ID NO: 3) is characterized by amino acid positions 171-325. In yet another embodiment, the DW domain of the FTPS isolated from Agrped1_689671 (SEQ ID NO: 2) is characterized by amino acid positions 170-324.
In a preferred embodiment, the mutation is selected from the group consisting of F204D, F204G, F204R, F2041, F204L and F204V of SEQ ID NO: 3.
In some embodiments, the genetically modified FTPS described herein is a linalool synthase, nerolidol synthase or both.
In one embodiment, the present invention refers to a genetically modified FTPS comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
In one aspect, the present invention refers to a method of producing a terpenoid comprising a) culturing the bacterial strain as described herein in an expression medium and b) isolating the terpenoid from said expression medium.
Culturing the bacterial strain in the expression medium will allow the expression of the one or more enzymes to produce one or more terpene precursors and expression of the FTPS.
In another aspect, the present invention refers to a method of producing a terpenoid comprising a) culturing a bacterial strain comprising a vector encoding the genetically modified FTPS as described herein in an expression medium and b) isolating the terpenoid from said expression medium.
The expression medium may be any culture medium that supports growth of the bacterial strain. The expression medium may comprise inducers capable of inducing the inducible promoter in the one or more vectors. The expression medium may also be an auto-inducing medium. In one example, the auto-inducing medium is ZYM5052. In other examples, the auto-inducing medium is lysogeny broth (LB), Terrific Broth (TB) or 2xPY.
The expression medium may be further supplemented with spherical C18 resin or Ni-nitrilotriacetic acid resin.
In one embodiment, the method described herein further comprises the step of isolating the FTPS from the bacterial cell and mixing the isolated FTPS with one or more terpene precursors to produce the terpenoid. The isolated FTPS may be mixed with the one or more terpene precursors in the same cell culture vessel or in a different vessel from the original culture. The FTPS may be isolated using a variety of methods. In some embodiment, the FTPS is isolated the bacterial cell by Ni-nitrilotriacetic acid resin, column based methods or both.
In another embodiment, the isolated FTPS described herein is further purified prior to mixing with one or more terpene precursors.
In yet another embodiment, the isolated FTPS is further mixed with one or more additional enzymes prior to mixing with one or more terpene precursors.
In one embodiment, the one or more additional enzymes described herein is Acetyl-CoA acetyltransferase, Hydroxymethylglutaryl-CoA synthase (HMGS), Hydroxymethylglutaryl-CoA synthase reductase (HMGR), IDI, melonate kinase (MK), Phosphomevalonate kinase (PMK) or mevalonate diphosphate decarboxylase (MVD1). In some embodiments, the Acetyl-CoA acetyltransferase is PhaA or atoB. [SF: any others?]
In one embodiment, terpenes or terpenoids may be produced using the FTPS of the present invention as follows: 1. Using crude cell lysate, the bacterial cells expressing only FTPS are harvested and lysed by freeze/thaw method and/or sonication method. The lysed cell supernatant containing soluble FTPS is mixed with substrates (GPP or FPP), 2.5 mM MgCl2 and 50 mM Tris/HCl buffer (pH 6.5-8.5) to produce terpenoids at 30-37° C. In another example, the FTPS will be purified from the bacterial cells by Ni-nitrilotriacetic acid resin and/or column-based method. The purified FTPS is mixed with substrates (GPP or FPP), 2.5 mM MgCl2 and 50 mM Tris/HCl buffer (pH 6.5-8.5) to produce terpenoids at 30-37° C. In addition, the FTPS may be coupled into a multienzyme reaction, for example at pH 7.5 and at 30° C., by mixing the FTPS with other enzymes such as IDI, MK, PMK or mevalonate pyrophosphate decarboxylase (PMD) to convert mevalonate into terpenoids.
In one embodiment, the product of the method described herein is a monoterpenoid, sesquiterpenoid or a mixture of both. In some embodiments, the monoterpenoid is selected from the group consisting of β-myrcene, linalool, geranyl acetate and combinations thereof.
In some embodiments, the sesquiterpenoid is selected from the group consisting of Δ6-protoilludene, α-muurolene, γ-muurolene, β-cadinene, β-copaene, δ-cadinene, δ-cadinene, epizonarene, α-cubebene, cubebol, epicubenol, nerolidol, viridiflorol, viridiflorene, α-cadinol, α-epi-cadinol, β-selinene, α-selinene, T-muurolol, β-elemene, β-gurjunene, germacrene A, germacrene D and combinations thereof.
In a preferred embodiment, the product is Δ6-protoilludene, linalool, geranyl acetate or combinations thereof.
The present invention also discloses the use of the FTPS described herein to produce one or more terpenoids.
In some embodiments, the one or more terpenoids is selected from the group consisting of β-myrcene, linalool, geranyl acetate, Δ6-protoilludene, α-muurolene, γ-muurolene, β-cadinene, β-copaene, δ-cadinene, γ-cadinene, epizonarene, α-cubebene, cubebol, epicubenol, nerolidol, viridiflorol, viridiflorene, α-cadinol, α-epi-cadinol, β-selinene, α-selinene, T-muurolol, β-elemene, β-gurjunene, germacrene A, germacrene D, trans-β-ocimene, β-cubebene, α-isocomene, longifolene, cadina-3,5-diene, caryophyllene, α-humulene, cubenene, calamenene, cubenol, δ-cadinol, cadina-1(6), 4-diene and combinations thereof.
In another embodiment, the one or more terpenoids described herein is produced in vitro or in vivo. In some embodiments, the one or more terpenoids is produced in vivo in a bacterial cell, a yeast cell, a plant cell, an animal cell or a fungal cell. In one example, the bacterial cell is an E.coli cell. In another example, the yeast cell is a Saccharomyces cerevisiae or a Yarrowia lipolitica cell.
In another embodiment, the one or more terpenoids described herein is produced in vitro.
In some embodiments, the FTPS described herein is isolated from a bacterial cell, a yeast cell, a plant cell, an animal cell or a fungal cell, and mixed with one or more terpene precursors to produce the one or more terpenoids.
In another embodiment, the isolated FTPS is further mixed with one or more additional enzymes prior to mixing with one or more terpene precursors. In one example, the one or more additional enzymes is IDI, MK, PMK or PMD.
The present invention also discloses a vector comprising a polynucleotide sequence encoding a 1-deoxyxylulose-5-phosphate synthase (DXS) enzyme comprising an amino acid sequence set forth in SEQ ID NO: 6, or a genetically modified 1-deoxyxylulose-5-phosphate synthase (DXS) enzyme as described herein.
In another example, the present invention refers to a vector comprising a polynucleotide sequence encoding a fungal terpene synthase (FTPS) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, or a genetically modified fungal terpene synthase (FTPS) as described herein.
The polynucleotide sequences generated from amino acid sequences may be optimized for improved expression in a host cell or an expression vector. The DNA sequences may be generated from amino acid sequences to have optimised Codon Adaptation Index (>0.6) and GC percentage (40-60%). Codon usage frequency table may be based on a strain of bacterial cell, for example, on Escherichia coli K-12 MG1655 strain. In most cases, a guided random method based on a Monte Carlo algorithm may be used. However, manual adjustments may be introduced to remove certain regions with complex secondary structures or repeated sequences. It will generally be understood that various codon optimization methods may be employed to improve expression of a protein or polypeptide in a host cell or expression vector.
In one aspect, the present invention refers to an FTPS encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.
In another aspect, the present invention refers to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:39.
In some embodiments, the nucleic acid sequence has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or, at least 99% or 100% identity to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.
The bacterial strain as disclosed herein may be used to characterize a FTPS. In another embodiment, the FTPS may be characterized by a product produced by the FTPS, the activity of the FTPS, or both.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
The fungal TPSs that have been cloned and functionally characterized are shown in Table 1.
Cultivation of Agrocybe aegerita and Analysis of its Fruiting Bodies
Agrocybe aegerita wildtype-strain AAE-3 was grown at 24° C. in the dark in modified crystallizing dishes (
Gas Chromatography-Mass Spectrometry Analysis of Terpenoids
Volatile compounds in the headspace were sampled at room temperature for 15 min by SPME with a DVB/CAR/PDMS (50/30 μm divinylbenzene/carboxen/polydimethylsiloxane) fiber (length 1 cm; Supelco, Steinheim, Germany). Compounds were desorbed in the split/splitless inlet (250° C. or 150° C.; SPME liner, 0.75 mm i.d.; Supelco) of an Agilent 7980B gas chromatography equipped with an Agilent 7200 accurate-mass quadrupole time-of-flight (GC/MS-TOF; Agilent Technologies, Singapore) for 1 min. In addition, for liquid culture analysis, dodecane (20% v/v) was used to extract the terpenoid produced in E. coli cultures. The obtained dodecane was diluted at 1:100 in hexane for GC-MS analysis. The GC/MS-TOF was equipped either with a VF-WAXms column (Agilent Technologies; 30 m×0.25 mm i.d., 0.25 μm film thickness) or a DB-5ms column (Agilent Technologies; 30 m×0.25 mm i.d., 0.25 μm film thickness), and the system was operated on the following conditions: (1) VF-WAXms, compounds were detected in split mode at split ratio of 10:1, the GC oven temperature was programmed to ramp from 80° C. (held for 2 min) to 240° C. (held for 5 min) at 10° C.×min−1; (2) DB-5ms, compounds were detected in split mode at split ratio of 10:1, the GC oven temperature was programmed to ramp from 50° C. (held for 2 min) to 160° C. at 10° C.×min−1, to 230° C. at 8° C.×min−1 and finally to 320° C. (held for 3 min). Mass spectra were acquired in the mass range of 33 300 m/z at the acquisition rate of 2 spectra/s. Ionization was performed by electron impact at 70 eV with an ion source temperature set at 230° C.
Structural Identification of Terpenoids
Mass spectra obtained by electron ionization mode were used for initial compound identification by comparing them with the spectra of terpenoids in the National Institute of Standards and Technology (NIST) database and published terpene spectra. Furthermore, Kovats retention indices of compounds produced were identified by calibrating with GC-MS with a C8-C30 alkane mix and were compared to the published retention indices in literature or in the NIST database. Major terpene products were verified, whenever possible, by comparison of retention time and mass spectra with authentic standards or essential oils with known terpene compositions. Niaouli essential oil [viridiflorene 6 (10.1% w/w), viridiflorol 7 (18.1% w/w)], Cedrela woods oil [α-muurolene 5 (1% w/w), δ-cadinene 4 (11.7% w/w)], Cubeb oil [germacrene D (1% w/w), γ-muurolene 2 (4.2% w/w), β-cubebene (4.4% w/w), cubebol (15.2% w/w)], Amyris wood oil [β-elemene (germacrene A) (0.1% w/w), δ-cadinenol, 0.2%]. In addition, the structure of Δ6-protoilludene was further confirmed by nuclear magnetic resonance spectroscopy.
Functional Annotation for Terpene Synthases and its Gene Clusters in the Agrocybe aegerita Genome
All the predicted amino acid sequences of protein-coding genes present in the genome of the dikaryotic strain A. aegerita AAE-3 have been searched for homologues to already characterized sesquiterpene synthases of Coprinopsis cinerea, Omphalotus olearius and Stereum hirsutum by blastp using Geneious® (version 9.1.8, Biomatters Ltd., Auckland, New Zealand). The predicted TPSs genes were then manually annotated. In addition, antiSMASH analysis was performed using the BiosynML plugin for Geneious® to predict terpene gene clusters in the A. aegerita genome.
Cloning and Expression of Terpene Synthase Genes in E. coli
Candidate fungal TPS genes were synthesized by Integrated DNA technologies and codon-optimized for expression in E. coli. The genes were cloned into pET11a vector for expression under the control of the T7 promoter. The resulting plasmid was transformed into BL 21 strains carrying the plasmid p15A-cam-T7-dxs-idi which was redesigned from the plasmid pACM-T7-dxs-T7-idi-T7-ADS-ispA. Furthermore, the dxs in the plasmid was mutated to SL3 or SL5 (
Homology Searches and Phylogenetic Tree Construction
The 11 A. aegerita TPSs were used to search other fungal TPSs in Basidiomycota and Ascomycota genomes sequenced and published by the Joint Genome Institute under the Fungal Genomics Program (http://genome.jgi-psf.org/programs/fungi/index.jsf) and in the UniProt database by Basic Local Alignment Search Tool program (http://www.uniprot.org/blast/). In addition, the previously published 392 basidiomycota TPSs were incorporated. The combined TPS candidates were manually inspected for duplicate sequences, erroneous protein predictions, such as incomplete sequences that deviated from the expected protein length (200-800 aa, except for two putative TPSs, Disac1_349444 and EXIGL_831178) or lacking the conserved metal-binding DxxxD and NSE/DTE triad, or with predicted additional domains (such as geranylgeranyl pyrophosphate synthase functions). Upon identification of putative TPS amino acid sequences, their alignments were performed using Clustal Omega and phylogenetic analyses were conducted with the Neighbor-Joining method using Clustal Omega or MEGA version 7.0.26.
Analysis of TPS Homologues by Sequence Similarity Networks
The curated fungal TPSs were analyzed by Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) web tool (http://efi.igb.illinois.edu/efi-est/) to generate sequence similarity networks (SSNs). The resulting SSNs were visualized using the open source software Cytoscape (http://www.cytoscape.org/). Inspection of the resulting SSNs is essential to obtain isofunctional clusters. Based on the SSNs generated by EFI-EST and sequentially varying a series of database-independent alignment score, a group of putative isofunctional groups (PIGs) were obtained. The data of PIGs and traditional phylogenetic trees were compared to select the putative isofunctional TPSs. Here, the three novel TPSs (viridiflorol synthase AAE3_13291, viridiflorene synthase AAE3_12839, and linalool/nerolidol synthase AAE3_9435) were chosen to probe other putative isofunctional TPSs which were further validated by experiments.
Δ6-Protoilludene Extraction and NMR Validation of its Structure
The AAE3_10454 recombinant E. coli strain was cultured in 200 mL of ZYM5052 auto-inducing medium, supplemented with 100 mg of the spherical C18 resin (VersaFlash spherical C18 bonded flash silica 45-75 um, Sigma-Aldrich). After 24 h of cultivation at 28° C. and 150 rpm, the cell culture was manually filtered by a C18 cartridge and was subsequently washed twice by deionized water. After filtration, the cells and liquid media were removed from the C18 cartridge. The terpene compound bound to the C18 resin was eluted by 10 mL of hexane. The eluted terpene solution was evaporated at 4° C. and subsequently analyzed on a Bruker DRX-400 NMR spectrometer with Cryoprobe, using 5-mm BBI (1H, G-COSY, multiplicity-edited G-HSQC, and G-HMBC spectra) or BBO (13C spectra) probe heads equipped with z-gradients. Spectra were calibrated to residual protonated solvent signals CHCl3 δH 7.24 and CDCl3 δC 77.23). The terpene compound was verified as Δ6-protoilludene by comparing the NMR spectral data with those reported in the literature.
Engineering an E. coli Strain for TPS Characterization.
The wild-type E. coli BL21 produces little amount of terpene precursors (GPP and FPP), therefore, it is not suitable as a TPS characterization platform. To improve the detection sensitivity and accuracy, DXS and IDI were overexpressed to improve the intracellular precursors (
Analysis of Terpenes Produced in A. aegerita
To obtain an estimate of terpenes produced in A. aegerita, volatile compounds produced by its liquid cultures were analyzed. The illudin precursor, Δ(6)-protoilludene 1, was a dominant metabolite produced by A. aegerita (
The Sesquiterpenome of A. aegerita
During fructification of A. aegerita 20 putative terpenoids were detected by means of GC/MS analysis, of which the tentatively identified Δ6-protoilludene# was the most prominent compound (
Characterization of 11 Predicted Sesquiterpene Synthases
All 11 predicted STSs were codon optimized and cloned into the pET vector, which was transformed into an engineered E.coli BL21 strain overproducing farnesyl pyrophosphate (FPP), the sesquiterpene precursor. Compounds tentatively identified on basis of their retention index (RI) and mass spectra in comparison to those in the literature and databases as described in the methods are marked with a hashmark (#).
All STSs (TPSs) except AAE3_09008 and AAE3_05024 gave rise to one or more sesquiterpenes in liquid cultures of the corresponding E. coli clone (
The E. coli strains expressing AAE3_0444 (SEQ ID NO: 27) and AAE3_6743 (SEQ ID NO: 29) produced several sesquiterpene compounds (
A wide variety of sesquiterpenes were detected for the E. coli culture expressing AAE3_09164 (SEQ ID NO: 30) (
The E. coli strain expressing AAE3_13190 (SEQ ID NO: 33) produced four major products, α-muurolene 5 (32%) and γ-muurolene 2 (22%), δ-cadinene 4 (24%) and δ-cadinol# 8 (21%) (
The major product of AAE3_12839 (SEQ ID NO: 34) was viridiflorene 6. In contrast, the E. coli strain expressing AAE3_13291 (SEQ ID NO: 32) produced viridiflorol 7 as the major product (viridiflorol 7 and viridiflorene 6 were confirmed by Niaouli essential oil,
Furthermore, the identified TPSs in A. aegerita shared the same first cyclization step with TPSs in C. cinerea and O. olearius. For Δ-6-protoilludene synthase (AAE3_4120 (SEQ ID NO: 26) and AAE3_10454 (SEQ ID NO: 31)), viridiflorene synthase (AAE3_12839 (SEQ ID NO: 34)) and viridiflorol synthase (AAE3_13291 (SEQ ID NO: 32)), they all proceed through 1,11 cyclization of FPP to form tricyclic sesquiterpenes (
Analysis of Fungal Genome for TPS Functional Study
The results in
With the 1408 TPSs, a phylogenetic tree which has seven major distinct TPS clades (
Predictive Framework to Uncover Other Fungal Viridiflorol Synthases
Knowledge acquired by studying the TPS products and the sequence conservation in each distinct clade provides a valuable basis for mechanistic understanding of the distinct activities. And it could be used to engineer and design more effective enzymes and to probe and even predict the functions of unknown TPSs. To test the predictive capability of the framework, identification of viridiflorol synthases in other species was carries out. The reason viridiflorol synthase was chosen is that there is only one type of plant viridiflorol synthase reported among all kinds of species. Analyzed by the phylogenetic tree in
Prediction and Validation of Fungal Linalool and Nerolidol Synthases (LNSs)
Starting with the sequence of AAE3_9435, identification of other NLSs in different fungal species was obtained by a BLAST search in databases of the Joint Genome Institute (JGI, http://jgi.doe.gov/fungi) and Universal Protein Resource (UniProt, http://www.uniprot.org/). EFI-EST analysis was carried out and a group of TPS homologues were shown to be clustered with AAE3_9435. By setting the alignment score to between 80 and 90, a smaller set of candidates were selected. With the selected cluster of TPSs in
Mutating the LS for a Different Function
As an exclusive monoterpene synthase, it was hypothesized that a point mutation of Agrped1_689675 (SEQ ID NO: 3) could change its function and products. To test this hypothesis, a few positions where Agrped1_689675 and the rest are different were highlighted (
The homologue model of Agr1 (Agrped1_689675) and Agr3 (Agrped1_689671) was built based on the structure of 1,8-cineole synthase from Streptomyces clavuligerus (PDB ID: 5nx5, 5nx6). The binding pocket, consisting of 15 residues within 6 Å from the substrate, was determined by PyMOL software v2.1.1 and highlighted here. The models were used to guide and understand the mutation of linalool/nerolidol synthases for improved selectivity or change of selectivity.
A summary of the sequence listing can be found in Table 5.
Agrocybe
aegerita FTPS
Agrocybe
pediades FTPS
Agrocybe
pediades FTPS
Galerina
marginata FTPS
Hypholoma
sublateritium FTPS
Escherichia
coli DXS
Solanum
lycopersicum FTPS
Melaleuca
quinquenervia FTPS
Galerina
marginata FTPS
Equivalents
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201807514P | Aug 2018 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2019/050438 | 9/2/2019 | WO | 00 |
