Patent Application
20230242944
Not Applicable
The contents of the electronic sequence listing (ISCA_041US.xml; Size 54,247 bytes; and Date of Creation: Jan. 30, 2022) is herein incorporated by reference in its entirety.
The present disclosure relates generally to a process for preparing insect pheromones in plants and microorganisms, and more particularly, insect pheromones of Diatraea saccharalis in plants and microorganisms.
The sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) is widely distributed throughout southern USA, Central America, and the tropical and subtropical zones of South America, it's dispersal likely mediated by human migration and trade. It's a key pest on sugarcane and maize and uses other grasses as hosts. The larvae cause damage by eating leaves and boring into stalks, which in sugarcane production where 19-25% of internodes are bored can diminish sugar yields by 8-20%. The estimated annual economic loss due to pest insects on sugarcane (in addition to D. saccharalis, other moth genera such as Chilo, Sesamia, and Scirpophaga also cause damage to sugarcane) is more than US$4.5 billion in Brazil alone, the world's largest sugarcane producer.
Insecticidal control of D. saccharalis is not efficient because the larval and pupal stages are protected inside the plant, and also because all developmental stages are present throughout the year, but integrated pest management (IPM), where the focus lies on monitoring, prevention and limited use of pesticides, optimised cultural practices using resistant cultivars, and biological control or the use of sex pheromones may provide a solution.
Pheromones are environmentally friendly alternatives to the use of traditional pesticides for the control of insect pests. For this purpose, synthetic pheromones are produced annually in large quantities. The use of pheromones for the control of pest insects has many advantages over the use of conventional chemical-based pesticides.
Pheromones are non-toxic. They have no adverse effects on non-target organisms, and do not kill parasitoids or other beneficial insects. The risks of resistance being developed in the pests are small. Even in terms of profit and reduction in damage, pheromones often compare favorably to the use of insecticides. In the case of treating cabbage against diamondback moth infestation, pheromone-based integrated pest management was found to be inexpensive ($62 relative to $123 per ha) resulting in a higher gross profit (ca $800 compared to $456 per ha) in comparison to the conventional practice with insecticides. The global market for pheromone-based control products is currently estimated to be approximately $200 million.
In 2010, around 40% of the sugarcane area in Brazil was treated by biological control, reducing pest damage by mass release of the larval parasitoid Cotesia flavipes (Hymenoptera: Braconidae) and the egg parasitoid Trichogramma galloi (Hymenoptera: Trichogrammatidae). The use of sex pheromones for monitoring or mating disruption has been successfully employed for other lepidopteran pests, though not yet for D. saccharalis, as field tests with the major sex pheromone component has shown low attractiveness to males compared to conspecific females.
The major female sex pheromone component of D. saccharalis has been identified as (9Z,11E)-hexadecadienal (Z9,E11-16:Ald), claimed in the U.S. patent application concerning the chemical synthesis of this compound to have been first reported by Hammond et al. at the meeting of Entomological Society of America in 1980. The first scientific publication identifying this pheromone component was in 2001 by Svatos̆ et al. In addition, three minor identified components, hexadecanal (16:Ald), (9Z)-hexadecenal (Z9-16:Ald) and (11Z)-hexadecenal (Z11-16:Ald), have been shown to elicit male antennal response and the more complex blends have been found to improve attraction of males in wind tunnel assays.
Studying how sex pheromones are biosynthesised by the insects is critical for development of biotechnological pheromone production to obtain the active compounds needed for pest management, and the successful use of insect enzymes for production of moth pheromones in yeasts and plant have been demonstrated. Moth sex pheromone biosynthesis involves genes in the large multigene families of fatty acyl desaturases (desaturases) and fatty acyl reductases (reductases), fatty alcohol oxidases and acetyltransferases. The biosynthesis of conjugated lepidopteran sex pheromones similar to Z9, E11-16:Ald has been described in moths from several families. The main pheromone components of Lampronia capitella (Prodoxidae) (9Z,11Z)-tetradecadienol (Z9,Z11-14:OH), Epiphyas postvittana (Tortricidae) (9E,11E)-tetradecadienyl acetate (E9,E11-14:OAc), and Spodoptera litura (Noctuidae) (9Z,11E)-tetradecadienyl acetate (Z9,E11-14:OAc) are made by one desaturase belonging to the clade of specific lepidopteran Δ11-desaturases, making both double bonds in sequence with a chain-shortening step in between. The major pheromone compounds of Cydia pomonella (Tortricidae) (8E,10E)-dodecadienol (E8,E10-12:OH) and Bombyx mori (Bombycidae) (10E,12Z)-hexadecadienol (E10,Z12-16:OH) are made by bifunctional desaturases that first introduce one double bond in the intermediate position and then turn this into the conjugated diene pheromone component. Two desaturases and several chain-shortening steps are involved in the biosynthesis of a pheromone component of Dendrolimus punctatus (Lasiocampidae), (5Z,7Z)-dodecadienol (Z5,Z7-12:OH), either using a desaturase homologous to common Δ9 acyl-CoA desaturases and a Lepidoptera specific Δ11-desaturase, or two Δ11-desaturases. The biosynthesis of the (7E,9Z)-dodecadienyl acetate (E7,Z9-12:OAc) pheromone component of Lobesia botrana (Tortricidae) involves a Δ11-desaturase and chain-shortening followed by the action of an elusive desaturase that makes a Δ7 double bond in the Z9-12:acyl. The biosynthetic pathways of these diunsaturated sex pheromone components may serve as hypotheses for D. saccharalis biosynthesis of Z9,E11-16:Ald.
As such, there is a need for improved methods with increased production of D. saccharalis pheromones and their precursors in plant or microbe factories.
The present invention demonstrates the feasibility of the production of large amounts of insect (moth) pheromone precursors of sugarcane borer DIATRAEA SACCHARALIS (Fabricius) (Lepidoptera: Crambidae).
In accordance with one aspect, the invention relates to relates to a genetically modified plant having incorporated into its genome a heterologous gene(s) encoding a first fatty-acyl desaturase, a second fatty-acyl desaturase, and a fatty-acyl reductase, wherein the plant produces at least one Diatraea saccharalis pheromone precursor.
In accordance with another aspect, the invention relates to a genetically modified microorganism having incorporated into its genome a heterologous gene(s) encoding a first fatty-acyl desaturase, a second fatty-acyl desaturase, and a fatty-acyl reductase, wherein the microorganism produces at least one Diatraea saccharalis pheromone precursor.
In accordance with yet another aspect, the invention relates to a method of producing Diatraea saccharalis pheromone precursors. The method involves selecting a plant or a microorganism to be genetically modified, incorporating into its genome, a heterologous gene encoding a first fatty-acyl desaturase, a second fatty-acyl desaturase, and a fatty-acyl reductase to obtain a genetically modified plant or a genetically modified microorganism, and producing Diatraea saccharalis pheromone precursors from the genetically modified plant or the genetically modified microorganism.
By way of this invention, it is for the first time that it has been made possible to produce Diatraea saccharalis pheromone precursors and therefrom Diatraea saccharalis pheromones.
These and other features and advantages of the various embodiments disclosed herein will be better understood with respect to the following description and drawings, in which like numbers refer to like parts throughout, and in which:
The detailed description set forth below is intended as a description of the presently preferred embodiment of the invention and is not intended to represent the only form in which the present invention may be constructed or utilized. The description sets forth the functions and sequences of steps for constructing and operating the invention. It is to be understood, however, that the same or equivalent functions and sequences may be accomplished by different embodiments and that they are also intended to be encompassed within the scope of the invention.
In the context of the present application and invention, the following definitions apply:
As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also, included are cell cultures of any species that can be cultured for the production of a chemical.
The term “genetic modification” implies the introduction of homologous and/or heterologous foreign nucleic acid molecules into the genome of a plant cell or into the genome of a microorganism, wherein said introduction of these molecules leads to an accumulation of insect pheromone precursors.
The term “recombinant microorganism” and “genetically modified microorganism” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous enzymes, to express heterologous enzymes, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene.
The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
The term “polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA.
The term “enzyme” as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides but can include enzymes composed of a different molecule including polynucleotides.
The term “exogenous” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
On the other hand, the term “endogenous” or “native” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
The term “heterologous” as used herein describes a relationship between two or more elements which indicates that the elements are not normally found in proximity to one another in nature. Thus, for example, a polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g., a genetically engineered coding sequence or an allele from a different ecotype or variety). An example of a heterologous polypeptide is a polypeptide expressed from a recombinant polynucleotide in a transgenic organism. Heterologous polynucleotides and polypeptides are forms of recombinant molecules.
The term “fatty acid” as used herein refers to a compound of structure R—COOH, wherein R is a C6 to C24 saturated, unsaturated, linear, branched, or cyclic hydrocarbon and the carboxyl group is at position 1.
The term “fatty alcohol” as used herein refers to an aliphatic alcohol having the formula R—OH, wherein R is a C6 to C24 saturated, unsaturated, linear, branched, or cyclic hydrocarbon.
The term “fatty acyl-CoA” refers to a compound having the structure R—(CO)—S—R1, wherein R1 is Coenzyme A.
In this disclosure, two plant platforms were utilized: Nicotiana benthamiana and Camelina sativa for pheromone production.
N. benthamiana is a close relative of N. tabacum, the most commonly grown commercial plant in the Nicotiana genus for its leaves to produce tobacco. Mature plants usually show a large variation in height, ranging from as tall as 1.5 meters to shorter than 200 mm. The Nicotiana species is favorable to work with in metabolic engineering aiming at production of pheromone compounds as they have relatively short production times, large area of leaves to output volatiles and are relatively easier to grow in controlled growth conditions. In addition, there is less concern about contaminating food supplies as they are not food crops.
Camelina was chosen as the oilseed production platform because it has limited use as a food crop and is considered an ideal system for rapid introduction and evaluation of fatty acid and other oil-related traits. Further, transgenes can easily be introduced into Camelina using a simple Agrobacterium-based method, and it has a relatively short life cycle that allows up to three generations in a year for evaluation of engineered traits. Camelina is also closely related to Arabidopsis thaliana, with a wealth of transgenic and genomic data for optimizing endogenous biosynthetic pathways for production of desired oil traits in seeds that typically are 30% to 40% oil by weight.
As discussed above, in a first aspect, the present invention relates to a genetically modified plant having incorporated into its genome a heterologous gene(s) encoding a first fatty-acyl desaturase, a second fatty-acyl desaturase, and a fatty-acyl reductase, wherein the plant produces at least one Diatraea saccharalis pheromone precursor. In an embodiment, the first fatty-acyl desaturase is a Δ9 desaturase and the second fatty-acyl desaturase is a Δ11 desaturase.
An exogenous fatty acyl desaturase described herein can be selected to catalyze the desaturation at a desired position on the hydrocarbon chain. Accordingly, in some embodiments, a Δ9 desaturase is capable of generating a double bond at C9 position and Δ11 desaturase at C11 position in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters.
The major female sex pheromone component of D. saccharalis has been identified as (9Z,11E)-hexadecadienal (Z9,E11-16:Ald), whereas the three minor components have been identified as hexadecanal (16:Ald), (9Z)-hexadecenal (Z9-16:Ald), and (11Z)-hexadecenal (Z11-16:Ald).
The present invention explores the production of pheromone precursors in their fatty alcoholic form which on oxidation can be readily converted to the aldehydes which are the pheromones.
In one embodiment, the first fatty-acyl desaturase and the second fatty-acyl desaturase together catalyze the conversion of a C16 fatty-acyl-CoA to at least one mono- or di-unsaturated C16 fatty-acyl-CoA.
In one embodiment, the first fatty-acyl desaturase and the second fatty-acyl desaturase generate a double bond at the C9 position and C11 position, respectively. In an exemplary embodiment, the first fatty-acyl desaturase is Dsac_KPSE (SEQ ID NO: 4), a desaturase obtained from D. saccharalis. In another exemplary embodiment, the second fatty-acyl desaturase is selected from Dsac_NPTQ (SEQ ID NO: 1), and Dsac_NPAQ, both desaturases obtained from D. saccharalis. In certain embodiments, the Dsac_NPAQ can be that from the published genome (Dsac_NPAQ genome), or can be the Dsac_NPAQ-end (SEQ ID NO: 2) or Dsac_NPAQ-start (SEQ ID NO: 3) disclosed herein, any of which can be referred to herein as Dsac_NPAQ.
In one embodiment, the fatty-acyl reductase catalyzes the conversion of the at least one mono- or di-unsaturated C16 fatty-acyl-CoA to at least one saturated, mono-, or di-unsaturated C16 fatty alcohol. In an exemplary embodiment, the fatty-acyl reductase is selected from Dsac_FAR_3781 (SEQ ID NO: 15).
The at least one saturated, mono- or di-unsaturated C16 fatty alcohol can be further oxidized to at least one saturated, mono-, or di-unsaturated C16 fatty aldehyde. Biosynthesis of pheromones using a genetically modified microorganism
In a second aspect, the present invention relates to a genetically modified microorganism having incorporated into its genome a heterologous gene(s) encoding a first fatty-acyl desaturase, a second fatty-acyl desaturase, and a fatty-acyl reductase, wherein the microorganism produces at least one Diatraea saccharalis pheromone precursor. In an embodiment, the first fatty-acyl desaturase is a Δ9 desaturase and the second fatty-acyl desaturase is a Δ11 desaturase.
In one embodiment, the first fatty-acyl desaturase and the second fatty-acyl desaturase together catalyze the conversion of a C16 fatty-acyl-CoA to at least one mono- or di-unsaturated C16 fatty-acyl-CoA.
In one embodiment, the first fatty-acyl desaturase and the second fatty-acyl desaturase generate a double bond at the C9 position and C11 position, respectively. In an exemplary embodiment, the first fatty-acyl desaturase is SEQ ID NO: 4, a desaturase obtained from D. saccharalis. In another exemplary embodiment, the second fatty-acyl desaturase is selected from SEQ ID NO: 1, and Dsac_NPAQ, both desaturases obtained from D. saccharalis.
In one embodiment, the fatty-acyl reductase catalyzes the conversion of the at least one mono- or di-unsaturated C16 fatty-acyl-CoA to at least one saturated, mono-, or di-unsaturated C16 fatty alcohol. In an exemplary embodiment, the fatty-acyl reductase is selected as SEQ ID NO: 15.
The at least one saturated, mono- or di-unsaturated C16 fatty alcohol can be further oxidized to at least one saturated, mono-, or di-unsaturated C16 fatty aldehyde.
In an embodiment, the microorganism is a yeast. In an exemplary embodiment, the yeast is Saccharomyces cerevisiae.
In a third aspect, the present invention relates to a method of producing Diatraea saccharalis pheromone precursors. The method involves selecting a plant or a microorganism to be genetically modified, incorporating into its genome, a heterologous gene encoding a first fatty-acyl desaturase, a second fatty-acyl desaturase, and a fatty-acyl reductase to obtain a genetically modified plant or a genetically modified microorganism, and producing Diatraea saccharalis pheromone precursors from the genetically modified plant or the genetically modified microorganism.
In an embodiment, the method involves catalyzing, by the first and the second fatty-acyl desaturases, conversion of a C16 fatty-acyl-CoA to at least one mono- or di-unsaturated C16 fatty-acyl-CoA. In another embodiment, the method involves catalyzing, by the fatty-acyl reductase, conversion of at least one mono- or di-unsaturated C16 fatty-acyl-CoA into at least one Diatraea saccharalis pheromone precursor. In yet another embodiment, the method further involves oxidizing the at least one Diatraea saccharalis pheromone precursor to at least one Diatraea saccharalis pheromone.
In different conditions, pheromones made using the invention's techniques and compositions including the pheromones can be employed to regulate Diatraea saccharalis insect behaviour and/or development. For instance, the pheromones can be employed to draw male Diatraea saccharalis insects to or away from a certain target region. Pheromones can be employed to draw Diatraea saccharalis insects away from agricultural regions that are particularly vulnerable. Insect monitoring, mass capturing, lure/attract-and-kill, or mating disruption strategies can all be utilised with the pheromones to draw in insects.
In accordance with the embodiments of the present invention, lures may be coated with, sprayed with, or otherwise impregnated with one of the pheromone compositions described in the current disclosure.
The pheromone compositions described in the disclosure may be employed in traps that are often used to draw Diatraea saccharalis insects. Such traps are widely utilised in many states and nations in pest eradication projects and are well recognised to those competent in the field. For retaining the pheromone mixture, the trap in one embodiment has one or more septa, containers, or storage receptacles. Thus, the current disclosure offers a trap that is loaded with at least one pheromone compound in certain embodiments. The pheromone compositions of the current disclosure can therefore be utilised in traps, for example, to entice Diatraea saccharalis insects as part of an approach for insect monitoring, mass trapping, mating disruption, or lure/attract and kill, for example, by incorporating a toxic substance into the trap to kill Diatraea saccharalis insects caught.
Pheromones made using the disclosed techniques can also be utilised to interfere with mating. Introducing artificial stimuli (such as the pheromone composition given here) that confuses the insects and disturbs mating location and/or courting prevents mating and stops the reproductive cycle. This approach is known as mating disruption and is used to control insect infestations.
The attract and kill approach, which can have the same results as mass-trapping, uses an attractant, such as a sex pheromone, to entice insects of the target species to an insecticidal chemical, surface, gadget, etc. for mass death and ultimate population reduction. When a synthetic female sex pheromone is used to attract male pests, such as moths, in an attract-and-kill technique, for example, a significant number of male moths must be killed over a prolonged period of time in order to restrict mating and reproduction and ultimately control the pest population.
In the following section, the aspect is described by way of examples to illustrate the processes of the invention. However, these do not limit the scope of the present invention. Several variants of these examples would be evident to persons ordinarily skilled in the art.
Reference chemicals for identification of pheromone gland (PG) compounds were purchased from Pherobank (Wijk bij Duurstede, The Netherlands), including hexadecanal (16:Ald), four isomers of hexadecenal (E/Z9-16:Ald and E/Z11-16:Ald) and the four isomers of (9,11)-hexadecadienal (Z9,E11-16:Ald). The aldehydes were used to prepare corresponding acids, methyl esters and alcohols according to previously reported protocols (Bjostad and Roelofs 1984; Corey and Schmidt 1979; Corso et al. 1998). Deuterium-labelled fatty acid for in vivo labelling, [16,16,16-2H3]-hexadecanoic acid (D3-16:acid), was purchased from Larodan (Malmo, Sweden), [16,16,16,15,15,14,14,13,13-2H9]-cis-11-hexadecenoic acid (D9-Z11-16:acid) and [8,8,7,7,6,6,5,5,4,4,3,3,2,2-2H14]-cis-9-hexadecenoic acid (D14-Z9-16:acid) from Cayman Chemicals (MI, USA). [16,16,16,15,15-2H5]-trans-11-hexadecenoic acid (D5-E11-16:acid) was prepared following Zarbin et al. (2007) (
Insects were procured from the Entomology Department of the Superior School of Agriculture Luiz de Queiroz at University of Sao Paulo, Brazil and reared in the lab on an artificial diet of soy flour, sugar and wheat germ, at conditions of 23° C., 70% relative humidity and a light:dark cycle of 16:8 h. Males and females were kept separately after the pupal stage. The time of dissection and number of sex pheromone glands (PGs) extracted for pheromone and total lipid/precursor analysis was based on pheromone amounts observed in the study by Batista-Pereira et al. (2002). Pheromone glands of 1 to 3 day old virgin females were dissected 3-5 h into scotophase and extracted in 15 μL heptane (Merck) per 5 glands. The solvent was transferred to a new vial after 15 min for pheromone analysis by GC/MS. For subsequent total lipid extraction and precursor GC/MS analysis the same sample was extracted again with chloroform:methanol (2:1 v/v) (Merck), overnight at room temperature, the solvent evaporated by a gentle stream of N2, and subjected to base methanolysis (Bjostad and Roelofs 1984) for transformation of lipids into fatty acid methyl esters (FAMEs). Identification of double-bond position of monounsaturated compounds was done using DMDS-adducts of FAMEs (Buser et al. 1983), produced by using 100 μL dimethyl disulfide (Merck) and 20 μL 5% I2 in diethyl ether (Merck), incubating at 40° C. overnight, then mixing with 50 μL 5% aqueous sodium thiosulfate (Merck) and 50 μL heptane.
Pheromone- and FAME PG extracts were analysed in split less mode using an Agilent 5975 mass detector (Agilent Technologies, Palo Alto, Calif., USA) coupled to an Agilent 6890 series gas chromatograph or an Agilent 5977B mass detector coupled to an Agilent 8890 series gas chromatograph, both fitted with an HP-INNOWax column (30 m×0.25 mm i.d., 0.25 m film thickness; J & W Scientific, Agilent Technologies, Santa Clara, Calif., USA). The GC inlet was set to 250° C. and the oven program to 80° C. for 1 min, increase of 10° C./min to 230° C., held for 10 min. The temperature of the transfer line was 280° C. and the MS source 230° C. DMDS-adducts were analysed using an Agilent 5975C mass detector coupled to an Agilent 7890A series gas chromatograph fitted with a HP-5MS column (30 m×0.25 mm i.d., 0.25 m film thickness, J & W Scientific, Agilent Technologies, Santa Clara, Calif., USA). The GC inlet was set to 260° C. and the oven program to 80° C. for 2 min, increase of 15° C./min to 140° C., then 5° C./min to 260° C. held for 15 min. The temperature of the transfer line was 280° C. and the MS source 230° C. Helium was used as the carrier gas.
Deuterium-labelled fatty acids D3-16:acid, D9-Z11-16:acid, D5-E11-16:acid, D14-Z9-16:acid and DMSO as a control were used for in vivo labelling, to monitor incorporation into the pheromone components and the potential pheromone precursors in the pheromone biosynthetic pathway. Labelled compound, 16 μg in a volume of 0.4 μL DMSO, was topically applied to the extruded female pheromone gland and abdominal tip 1 h before extraction of the pheromone gland. Pheromone gland extraction and pheromone and precursor analyses by GC/MS were performed as described above.
Thirty female PGs and 32 male abdominal tips for transcriptome analysis were dissected as described above and stored immediately at −80° C. prior to RNA extraction. RNA extraction was done using TRIzol reagent (Thermo Fisher) and RNA cleanup and concentration using the RNeasy Micro kit (QIAGEN), both steps following the manufacturers' instructions. The RNA concentration was measured using a 2100 Bioanalyzer system (Agilent). Library preparation with Illumina TruSeq poly-A enrichment and 150 bp paired-end Illumina sequencing using a NovaSeq6000 system was done by SciLifeLab National Genomics Infrastructure (Stockholm, Sweden), for one female and one male replicate. FastQC v0.11.5 (bioinformatics.babraham.ac.uk/projects/fastqc) was used to assess quality of the reads, and low quality raw reads were filtered and adaptors removed using Trimmomatic v0.36 (Bolger et al. 2014) and Prinseq v0.20.4 (Schmieder and Edwards 2011). Assembly was done using the Trinity software package v2.8.2 (Grabherr et al. 2011; Haas et al. 2013) with default parameters except normalize_max_read_cov 50—min_kmer_cov 2—min_glue 2—KMER_SIZE 23, and completeness of the assembly assessed with BUSCO v3.0.2b and the Insecta- and Endopterygota datasets (https://busco.ezlab.org/). TransDecoder v5.0.1 (github.com/TransDecoder/) was used to extract ORFs and predict protein coding regions, and differential expression between female and male tissues estimated with RSEM v1.3.1 (Li and Dewey 2011) and Trinity package scripts align_and_estimate_abundance.pl and abundance_estimates_to_matrix.pl. Fatty acid desaturases were found using the FA_desaturase (PF00487) family of the Pfam domain database (Mistry et al. 2020) together with HMMER v3.2.1 (hmmer.org), and fatty acid reductases and fatty alcohol oxidases were found by BLAST (Altschul et al. 1990) homology search with other lepidopteran sequences. For phylogenetic analyses, D. saccharalis desaturases and reductase amino acid sequences, together with other lepidopteran sequences available from GenBank (ncbi.nlm.nih.gov), were aligned using MAFFT (Katoh et al. 2002) and scoring matrix BLOSUM62, and maximum-likelihood phylogenies were constructed with FastTree v7.450 (Katoh et al. 2002; Katoh and Standley 2013) and visualised with FigTree v1.4.4 (github.com/rambaut/figtree/).
cDNA was synthesised from the PG RNA sample with the Thermoscript RT-PCR kit (Thermo Fisher) following the manufacturer's protocol. To verify transcript sequences, all full-length gene ORFs were amplified and Sanger sequenced. For yeast episomal expression, desaturase—and reductases were cloned using Gateway technology (Thermo Fisher) into pDONR221 followed by pYEX-CHT (Patel et al. 2003) or pYES-DEST52 vectors, and transformed into the yeast strain Δole1/Δelo1 (MATa elo1::HIS3 ole1::LEU2 ade2 his3 leu2 ura3) (Schneiter et al. 2000) or INVSc1 (MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52) (Thermo Fisher) using the S.c. EasyComp transformation kit (Thermo Fisher) following the manufacturers' protocols. Uracil prototrophs were selected for and cultivated in medium containing 1.92 g/L dropout medium lacking uracil (Formedium), 6.7 g/L yeast nitrogen base (Merck), 0.08 g/L adenine (Merck), 1.5% tergitol (Merck), 100 mM oleic acid (Merck) and 2% glucose (Merck) or 2% galactose (Merck) and 1% raffinose (Merck). The medium also contained 1 mM 12:Me and 14:Me, except for expressions of desaturases SEQ ID NO: 1, Dsac_NPAQ and SEQ ID NO: 4. A 24-72 h pre-cultivation was followed by 48-72 h incubation at 30° C. of 4-50 mL cultures, where the heterologous gene expression was induced with a final concentration of 0.5-1 mM CuSO4 (Merck) and/or 2% galactose. Cells were harvested and subjected to total lipid GC/MS analysis as described above, or as follows: extraction with 1 mL methanol:chloroform (2:1, v/v) and 1 mL 0.075 M acetic acid (Merck), followed by running the organic phase on a silica gel TLC plate (Silica gel 60, Merck) that was developed in heptane:DEE:HAc (85:15:1,v/v/v), the bands visualized by spraying water and target gel areas collected separately into 4 mL vials and extracted with 1 mL methanol:chloroform (2:1, v/v) in a sonication bath for 2 min. The extract was then centrifuged at 2,000 g for 1 min. The supernatant was transferred to a new vial and 1 mL 0.075 M acetic acid was added to partition the lipids into chloroform. The chloroform phase containing alcohols was transferred into a new vial and evaporated to dryness, followed by addition of 40 μL heptane and GC/MS analysis as described above. For yeast integrative expression, constructs were cloned by fusion PCR and Gateway technology into vector pCfB2875, modified from Maury et al. (2016), and transformed into INVSc1 and cultivated as described above, in 25-100 mL and for four days. Total broth or only supernatant was extracted using 2 mL heptane/25 mL culture and analysed by GC/MS as described above.
Extracts of pheromone glands (PGs) contained the previously identified sex pheromone components of D. saccharalis (16:Ald, Z9-16:Ald, Z11-16:Ald and Z9,E11-16:Ald) and their corresponding fatty acid precursors (
For elucidation of the pathway for sex pheromone biosynthesis, deuterium-labelled potential pheromone precursors were used in in vivo labelling experiments (
Transcriptome sequencing yielded 390 M raw reads per sample, and 282-302 M reads per sample after quality filtering. The assembly using both female and male samples resulted in 267,839 total assembled transcripts with an average length of 831 bp, N50 length of 1,689 bp and an overall alignment rate of 77%. Looking at ExN50 statistics, 25,333 transcripts are covered by 83% of reads and N50 length of this subset is 2,360 bp (
The transcriptome assembly was searched for putative desaturase, reductase and alcohol oxidase genes involved in sex pheromone biosynthesis, and 12, ten and one full-length genes, respectively, were found by homology search with known lepidopteran genes (Table 1 and transcript assembly nucleotide sequences). For four of these genes the expression level was considered both female-biased (expression ratio female:male >10) and relatively high (>100 TPM) (
Desaturases and reductases for which expression in the transcriptome analysis was considered both female-biased (female:male >10) and relatively high (>100 TPM), and all remaining full-length desaturases found in the transcriptome, were together with the desaturase Dsac_NPAQ from the published genome (Borges dos Santos et al. 2020), functionally characterised in a yeast expression system. After expression, their products were methylated and analysed by GC/MS (
We have shown that biosynthesis of the major D. saccharalis sex pheromone component, Z9,E11-16:Ald, involves two different desaturases acting in sequence, the Δ9 desaturase SEQ ID NO: 4 acting on palmitic acid and producing Z9-16:acid, followed by the Δ11 desaturation by SEQ ID NO: 1 to produce Z9,E11-16:acid (
The presented pathway is supported by the presence of 16:acid and Z9-16:acid precursors in the PG while no E11-16:acid or E/Z10-16:acid was seen. This indicates that saturated 16:acid is the precursor being desaturated (as opposed to for example 18:acid), and that the Z9 double bond in the diene is made first, instead of the E11 double bond or the Z9,E11 double bond being made by a bifunctional desaturase from a Δ10 double bond. In vivo labelling showed that 16:acid and Z9-16:acid precursors were incorporated into the pheromone component whereas E11-16:acid was not. The functional assay of the desaturases in the yeast background deficient in native Δ9 desaturation activity showed that SEQ ID NO: 4 displays broad Δ9 desaturation activity with a preference for producing Z9-16:acid, the precursor of Z9,E11-16:Ald and also the minor pheromone component Z9-16:Ald. SEQ ID NO: 1 displays Δ11 desaturation activity to produce the major pheromone component precursor, and the other three isomers, Z9,Z11-16:acid, E9,E11-16:acid and E9,Z11-16:acid. It also produces the precursor of pheromone component Z11-16:acid. This type of desaturation pathway is similar to that seen for the pine caterpillar moth D. punctatus, where a Δ9 (Dpud9_KPSE) and a Δ11 (Dpud11_LPAE) desaturase are involved in the biosynthesis of a diene pheromone component precursor Z5,E7-12:acid (Liénard et al. 2010). Our in vivo labelling experiments could establish that Z9-16:acid is the precursor of two of the dienes isomers seen in the PG, Z9,E11-16:acid and Z9,Z11-16:acid, while no incorporation was seen into the two isomers with an E9 double bond. Possibly these come from the E9-16:acid precursor produced by SEQ ID NO: 4.
The pgFAR SEQ ID NO: 15 was in the yeast assay shown to have a broad specificity and ability to reduce all the D. saccharalis pheromone precursor components into their respective alcohols. This pgFAR and the two described desaturases being part of the pheromone biosynthesis pathway is corroborated by their high expression levels and female PG biased expressions. Even if mRNA expression level does not always have an obvious biological significance and is not an undisputable indicator of protein expression level (Koussounadis et al. 2015), transcript expression level and PG bias has in sex pheromone biosynthesis studies so far been a good indicator for involvement in the pathway (Antony et al. 2015; Strandh et al. 2008; Zhang et al. 2014). A less expressed gene candidate might be involved, but often this lower expression represents a suboptimal sampling time or less discriminate tissue dissection. A putative fatty alcohol oxidase (FAO) responsible for the last step in D. saccharalis pheromone biosynthesis, namely conversion of the alcohol precursors into their aldehyde pheromone counterparts, was identified in the transcriptome based on similarity to other putative moth FAOs (Xia et al. 2022), its high expression level and female PG bias. An assay for functional characterisation of a FAO or other aldehyde oxidases by heterologous expression in yeast and monitoring of aldehyde production has not yet been established and may be difficult due to the toxicity of aldehydes in such a system.
An unambiguous Dsac_NPAQ transcript could not be inferred from the de novo transcriptome assembly, and neither could a transcript identical to the one ultimately amplified from cDNA, SEQ ID NO: 1. The completeness of the assembly was assessed to be high at 94% when comparing to the BUSCO dataset of conserved Insecta genes. Ambiguities in transcriptome assemblies can be related to allele variation or gene families (paralogs), which could explain the difficulty observed in resolving this sequence that possibly correspond to two highly similar transcripts (Razo-Mendivil et al. 2020). Lepidopteran desaturases are part of a large gene family of both ancient and more recent duplicated and evolved genes (Lienard et al. 2008). The ambiguous sequence might be two very similar genes merged because of low stringency in assembly parameters, creating fragmented or chimeric sequences. Sometimes fragmented sequences can be completed by increasing the k-mer size in the assembly parameters, but highly similar transcripts resulting in chimeras (or lowly expressed paralogs, with low coverage) might possibly only be resolved by lowering the k-mer size, which in turn can result in a highly fragmented assembly (Gruenheit et al. 2012). If the consensus sequence for Dsac_NPAQ is chosen as the transcript assembly most similar to Dsac_NPAQ from the available D. saccharalis genome assembly (Borges dos Santos et al. 2020), it's 17 amino acid residues different from the SEQ ID NO: 1 sequence amplified from cDNA. It might not be possibly to resolve two such highly similar sequences with similar expression levels, by short-read transcriptome sequencing and de novo assembly. Two such sequences would be more easily resolved if one of them had lower expression, and then a certain coverage cut-off level could be used to unambiguously assemble only the sequence with high expression level (Gruenheit et al. 2012). The D. saccharalis genome assembly presented in Borges dos Santos et al. (2020) is 87% complete assessed by the BUSCO Insecta dataset, and does resolve two full-length genes, Dsac_NPAQ (genome) and SEQ ID NO: 1 (genome), differing in 35 amino acid residues and also in their intron sequences. These are highly similar to Dsac_NPAQ from this study's transcriptome assembly and SEQ ID NO: 1 amplified from the transcriptome cDNA, respectively. These could represent different alleles (Lassance et al. 2010), or a recent duplication event creating two paralogs that are still both functional and exhibit high expression levels, where one might evolve to become redundant in future pheromone biosynthesis of D. saccharalis. It could also be that both paralogs contribute to the specificity of the D. saccharalis pheromone blend. Both versions of this desaturase expressed in yeast in this study (SEQ ID NO: 1 (cDNA) and Dsac_NPAQ (genome)) were functionally capable of producing the pheromone component precursors Z11-16:acid and Z9,E11-16:acid (and its isomers), although at different ratios. These sequences differ in 33 amino acid residues, possibly revealing positions in the primary sequence that are functionally redundant, but also responsible for the ratio difference. 30 out of the 33 are considered conservative substitutions, and none are within domains characterised as important for the catalytic function, such as the conserved histidine-residues, the CoA-binding site or the coordinating carboxylates (Bai et al. 2015). 14 substitutions are within either trans-membrane domains or alpha-helices as characterised in the crystal structure of the mammalian stearoyl-CoA desaturase in Bai et al. (2015), and 19 are in domains not structurally or functionally defined.
The alcohol precursor titres presented in this study are too low to be immediately relevant for industrial biotechnological production and use in prospective IPM applications. Expression of SEQ ID NO: 4, 2× SEQ ID NO: 1/NPAQ and SEQ ID NO: 15 produced a titre of only 2.7±0.4 mg/L Z9,E11-16:OH precursor in the broth of small-scale cultivations. Before a large-scale yeast production of D. saccharalis pheromone precursor(s) can be initiated, using the identified biosynthetic pathway, it's relevant to optimise with regard to yeast host, platform strain and fermentation conditions, possibly even enzyme engineering for more active and specific desaturases. Some of these optimisations may involve manipulating the flux in the yeast fatty acid biosynthesis pathways, the copy number of pheromone biosynthesis genes and eliminating production of unwanted side-products (Holkenbrink et al. 2020). A biotechnological production could consist of heterologous synthesis of all the pheromone component precursors at a biologically relevant ratio, for trapping and monitoring purposes, but this would be difficult. The ratios of pheromone component precursors produced in yeast in this study are much different from what is seen in the D. saccharalis females, differences that are expected considering the two very different biological systems and their possibly incomparable gene expression levels. It would be most efficient to develop biotechnological production of the major and also most complex structure of the blend components, for possible use in mating disruption. The minor components might be produced more easily by already published biological production platforms, especially 16:acid and Z9-16:acid as they are native products in relevant heterologous platforms (Holkenbrink et al. 2020; Xia et al. 2020). A platform for production of Z9,E11-16:OH alone would facilitate down-stream processing, where the diene could be purified from saturated—and monounsaturated products by urea-complexation (Hayes et al. 1998). It would be difficult to isolate Z9,E11-16:acid from its isomers, and there are many examples of geometric isomers or pheromone analogues that disrupt pheromone communication by acting as behavioural antagonists (Eizaguirre et al. 2007; Juárez et al. 2016; Wang et al. 2022; Witzgall et al. 1999). Purification from the isomers might not be necessary for D. saccharalis IPM, as they don't elicit any antennal responses in GC-EAD experiments (Batista-Pereira et al. 2002).
Despite the observed deviations of the yeast extract from an optimal D. saccharalis pheromone blend, it might still be active for IPM purposes, as seen for a crude extracts of the codling moth (Cydia pomonella) pheromone produced by engineered oilseed plants Camelina sativa (Xia et al. 2021). Here traps with crude pheromone plant extract still caught males, although not at levels of purified extracts or synthetic pheromone. Field tests with sticky traps baited with live D. saccharalis females were already in the 1960s shown to reduce the damage on sugarcane in Louisiana (Hammond and Hensley 1971). However, it has also been shown in the field that traps baited with Z9,E11-16:Ald alone is much less attractive to males compared to live females (Svatos̆ et al. 2001), and the newly identified four-component blend (da Silva et al. 2021) has shown similar inferior attractiveness (P. H. G. Zarbin, unpublished data). Research on optimisation of component ratio and dosage in trap lures could improve attractiveness in the field. The low trap attractiveness could also be explained by a pheromone blend that is still incomplete, or enhancing or synergistic effects of compounds present in the gland extract (Hall et al. 2017; Meier et al. 2016) that don't necessarily elicit detectable antennal responses in GC-EAD analyses for this species. Pheromone blend components could remain elusive because this species show low electrophysiological responses to minor pheromone components, as observed by Batista-Pereira et al. (2002) to Z11-16:Ald, and also by Kalinova et al. (2005), where >100 ng was needed for a significant response as opposed to <1 ng of Z9,E11-16:Ald. Biosynthesis studies such as the present could aid in elucidation of putative missing blend components, in the form of specific precursors observed in the pheromone gland and/or genes showing female-biased expression and a specialised function or producing minor by-products related to the major pheromone components. Further studies will have to be carried out to reveal any unknown components in the pheromone blend, compounds that are possibly produced in very low quantities in the pheromone gland but may be crucial in eliciting male attraction to a synthetic blend comparable to that of conspecific females.
Mating disruption has not been assessed for this species, and might still work relying on only the major pheromone component, as seen for other lepidopteran pests, for example the European grapevine moth (Lobesia botrana) (Ioriatti et al. 2011). Mating disruption based on biotechnologically produced sex pheromones, in a green and cheap production, can be the way forward in controlling D. saccharalis in the vast Brazilian sugarcane fields. Half of the cultivated land area in Brazil is covered by sugarcane crops (FAOSTAT 2021; Parra 2014), and it's a crop important for both sugar- and ethanol industries, smallholder farmers and as a renewable biomass (da Cunha Borges Filho et al. 2019; Dias and Sentelhas 2018; Goebel and Sallam 2011). Because D. saccharalis damage has a significant economic impact and is difficult to control using conventional insecticides, it's pertinent and pressing to develop IPM technologies to control this pest.
The above description is given by way of example, and not limitation. Given the above disclosure, one skilled in the art could devise variations that are within the scope and spirit of the invention disclosed herein, including various variations of Dsac_NPAQ. Further, the various features of the embodiments disclosed herein can be used alone, or in varying combinations with each other and are not intended to be limited to the specific combination described herein. Thus, the scope of the claims is not to be limited by the illustrated embodiments.
This application claims the benefit of U.S. Provisional Application No. 63/305,088, filed on Jan. 31, 2022, the teachings of which are expressly incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63305088 | Jan 2022 | US |
