WO2024253742A1 - Engineering human skin microbes to produce mosquito repellent terpenes - Google Patents

Engineering human skin microbes to produce mosquito repellent terpenes Download PDF

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WO2024253742A1
WO2024253742A1 PCT/US2024/023221 US2024023221W WO2024253742A1 WO 2024253742 A1 WO2024253742 A1 WO 2024253742A1 US 2024023221 W US2024023221 W US 2024023221W WO 2024253742 A1 WO2024253742 A1 WO 2024253742A1
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bacterial cell
seq
engineered bacterial
acid sequence
amino acid
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Mohamed Ibrahim HASSAN
Christopher A. Voigt
Geng-Min Lin
Daniel Yoo
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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Definitions

  • Mosquitoes are a major source of vector-bome diseases including malaria, dengue, West Nile, zika and yellow fever, affecting millions every year.
  • Vector-bome diseases account for 17% of all infectious diseases, resulting in >700,000 deaths annually (World Health Organization, 2022).
  • Minimizing contact of mosquitoes is key to preventing the transmission of illnesses.
  • the disclosure provides engineered bacterial cells for producing a mosquito-repelling terpene, comprising an engineered terpene biosynthetic pathway introduced into a bacterial cell that is not otherwise capable of producing the terpene, wherein the engineered bacterial cell is capable of producing the mosquito-repelling terpene.
  • the bacterial cell is a human skin microbe; in other embodiments, the bacterial cell is Corynebacterium amycolatum, Staphylococcus caprae. or Staphylococcus epidermidis.
  • the mosquito-repelling terpene is selected from the group consisting of eucalyptol, kaur-16-ene, and kolavelool. In one embodiment, the mosquitorepelling terpene comprises eucalyptol. In one such embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic
  • AtoB acetyl-CoA C-acetyltransferase
  • HMGS hydroxymethylglutaryl-CoA synthase
  • HMGR hydroxymethylglutaryl-CoA reductase
  • MK mevalonate kinase
  • PMK phosphomevalonate kina
  • the mosquito-repelling terpene comprises kaur- 16-ene.
  • the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: wherein the bacterial cell is transformed with polynucleotides encoding the folio-wing polypeptides having enzymatic activity: acetyl- CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS), PtmT2 cyclase (CycA07), and Hypo
  • the mosquito-repelling terpene comprises kolavelool.
  • the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS); TR51 15710 cyclase (CycAOl); and terpene synthase family (CycBOl).
  • AtoB acetyl-CoA C-acetyltransferase
  • the disclosure provides polynucleotides comprising the nucleotide sequence of any one of SEQ ID NO: 13, 17, and 20, recombinant expression vector comprising the polynucleotides, and recombinant host cells compnsing the polynucleotide or recombinant expression vector.
  • compositions comprising a plurality of the engineered bacterial cell of any embodiment or combination of embodiments herein, and a pharmaceutically acceptable carrier.
  • the compositions are formulated for topical administration, including but not limited to a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
  • the disclosure provides methods to repel mosquitos, comprising contacting the skin of a subject with a plurality of the engineered bacterial cells of any preceding claim, or with the composition of any embodiment herein, thereby repelling
  • SUBSTITUTE SHEET (RULE 26) mosquitos from skin of the subject.
  • the disclosure provides methods to repel mosquitos, comprising contacting the skin of a subject with kaur- 16-ene, kolavelool, or a combination thereof, thereby repelling mosquitos from skin of the subject.
  • FIG. 1 Terpene production pipeline to produce potential mosquito repellents.
  • A Outline of E. coli strain harboring biosynthetic pathway including the mevalonate pathway, a prenyl transferase and terpene cyclase genes for in vivo terpene production. This strains are cultivated in screw cap vials and analyzed by GC-MS for terpene production.
  • B Select list of terpenes produced in E. coli.
  • FIG. 1 Mosquito olfactometer set up to determine repellent terpenes
  • A Overview of mosquito olfactometer. Mosquitoes are placed in the start canister prior to the initiation of the experiment. A petri dish with 1 mL of bacterial culture (after cultivation for 24 h) is placed in the stimulus box. Repellency is determined by the number of mosquitoes travelling from the start canister, through the flying tube and ultimately residing in the trap after 5 minutes.
  • B E. coli strains producing eucalyptol and kaur- 16-ene repel mosquitoes (box) when compared to an E. coli control.
  • LA lactic acid + ammonia are used to stimulate mosquito response in the olfactometer.
  • FIG. 3 Engineering of a primary skin isolate to repel mosquitoes.
  • A Engineered S. epidermidis LM088 (sMIH197) to contain a constitutively expressed biosynthetic pathway of eucalyptol on the genome.
  • B Engineered strain successfully produces eucalyptol, as detected by GCMS.
  • C SMIH197 repels mosquitoes when analyzed in mosquito olfactometer described in Figure 2A.
  • Figure 4 Engineenng of a skin isolate to produce repellent kaur-16-ene.
  • A Biosynthesis of kaur- 16-ene utilizes two diterpene cyclases (cycA07, cycB04). Successfully engineered S. epidermidis LM088 (sMI210) to constitutively express kaur-16-ene pathway.
  • B Engineered strain successfully produces kaur- 16-ene, as detected by GCMS.
  • FIG. 1 Engineering of a primary' skin isolate produce Kolavelool. Biosynthesis of kolavelool utilizes two diterpene cyclases (cycAOl, cycBOl). Successfully engineered A epidermidis LM088 (sMI217) to constitutively express kolavelool pathway. (B) Engineered strain successfully produces kolavelool, as detected by GCMS
  • the disclosure provides engineered bacterial cells for producing a mosquito-repelling terpene, comprising an engineered terpene biosynthetic pathway introduced into a bacterial cell that is not otherwise capable of producing the terpene, wherein the engineered bacterial cell is capable of producing the mosquito-repelling terpene.
  • the inventors have identified mosquito- repellant terpenes and heterologously expressed terpene biosynthetic pathways in human skin microbes that can successfully generate the mosquito-repellant terpenes.
  • mosquito-repellant terpene means a terpene that deters mosquitoes from approaching or settling on a subject.
  • the engineered bacterial cells may be any bacteria that is not otherwise capable of producing the mosquito-repellant terpene.
  • the bacterial cells are human skin microbes. Many such human skin microbes are known and included in the present disclosure.
  • the bacterial cell may be a primary isolate from human skin.
  • the bacterial cell may be one that is available from a depository or commercially.
  • the bacterial cells comprise Corynebacterium amycolatum, Staphylococcus caprac. or Staphylococcus epidermidis. Exemplary sources for such cells include, but are not limited to, primary isolates isolated using standard techniques
  • SUBSTITUTE SHEET (RULE 26) (including but not limited to Staphylococcus epidermidis LM088), Corynebacterium amycolatum ATCC 49368, Staphylococcus caprae ATCC 55133, and Staphylococcus epidermidis ATCC 12228.
  • the mosquito-repelling terpene is selected from the group consisting of eucalyptol, kaur-16-ene, and kolavelool.
  • the structures of these compounds are shown below.
  • the mosquito-repelling terpene comprises eucalyptol.
  • the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranyldiphosphate synthase (GPPS), and 1,8 cineole synthase (bCinS).
  • a list of the genes and encoded proteins are shown in Tables 1-2, and further details are provided in the examples.
  • Table 1 List of genes for the biosynthesis of Eucalyptol
  • the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of
  • SEQ ID NO: 2 hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO: 5, diphosphomevalonate decarboxylase (MVD) comprising the ammo acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta-isomerase (IDI) comprising the amino acid sequence of SEQ ID NO:7, geranyldiphosphate synthase (GPPS) comprising the amino acid sequence of SEQ ID NO:8, and 1,8 cineole synthase (bCinS) comprising the amino acid sequence of SEQ ID NO:9.
  • HMGR hydroxymethylglutaryl-CoA reductase
  • MK mevalonate kinase
  • PMK phosphomevalon
  • the polynucleotides may be codon-optimized for expression in the bacterial cell of interest.
  • the bacterial cell may be transfected with separate polynucleotides encoding each polypeptide, with a single polynucleotide encoding all of the polypeptides, or 2 or more polynucleotides each encoding 1 one or more of the polypeptides.
  • the polynucleotides are arranged in a single operon, with all polypeptides encoded by a single polynucleotide.
  • the polynucleotides encoding the different polypeptides may be in any order in the operon.
  • the polynucleotide encoding bCinS is 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity.
  • the operon comprises, in 5’ to 3’ order, polynucleotides encoding bCmS, atoB, HMGS, HMGR,MK, PMK, MVD, idi, and gpps.
  • the operon comprises a heterologous promoter 5’ to the polynucleotides encoding the polypeptides. In one such embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotide bCinS.
  • heterologous promoter As used throughout herein, a “heterologous” promoter, insulator, or ribosome binding site is one that not naturally occurring as a regulatory' sequence for the genes it is controlling expression of. Any heterologous promoter may be used as appropriate for an intended purpose.
  • the heterologous promoter comprises the nucleotide sequence of SEQ ID NOTO, 21, 22, 24, or 25. In one embodiment, the heterologous promoter comprises the nucleotide sequence of SEQ ID NOTO.
  • the operon comprises a heterologous insulator 5’ to the polynucleotide sequence encoding bCinS.
  • the heterologous insulator is 3’ to the heterologous promoter. Any heterologous insulator may be used as appropriate for an intended purpose.
  • the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23.
  • the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11.
  • the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 9 of the genes encoding the polypeptides. Any heterologous RBS may be used as appropriate for an intended purpose.
  • the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
  • the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13, as shown below.
  • SUBSTITUTE SHEET (RULE 26) 3301 caggagcagt ctctgaattt tattcaggcc aacttgttgc aggttatgat aaaatgttaa
  • the mosquito-repelling terpene comprises kaur- 16-ene.
  • the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: wherein the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl -Co A C -acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD),
  • SUBSTITUTE SHEET (RULE 26) Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS), PtmT2 cyclase (CycA07), and Hypothetical cyclase (CycB04).
  • IDI Isopentenyl-diphosphate Delta-isomerase
  • GGPS geranylgeranyl pyrophosphate synthase
  • PtmT2 cyclase (CycA07)
  • Hypothetical cyclase (CycB04).
  • the bactenal cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO:1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of
  • SEQ ID NO: 2 hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO: 5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta-isomerase (IDI) comprising the amino acid sequence of SEQ ID NOT; geranylgeranyl pyrophosphate synthase (GGPS) comprising the amino acid sequence of SEQ ID NO: 14; PtmT2 cyclase (CycA07) comprising the amino acid sequence of SEQ ID NO: 15; and Hypothetical cyclase (CycB04) comprising the amino acid sequence of SEQ ID NO: 16.
  • HMGR hydroxymethylg
  • the bactenal cell may be transfected with separate polynucleotides encoding each polypeptide, with a single polynucleotide encoding all of the polypeptides, or 2 or more polynucleotides each encoding 1 one or more of the polypeptides.
  • the polynucleotides are arranged in a single operon, with all polypeptides encoded by a single polynucleotide.
  • the polynucleotides encoding CycA07 and CycB04 are 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity.
  • the operon comprises, in 5’ to 3’ order, polynucleotides encoding CycA07, CycB04, atoB, HMGS, HMGR, MK, PMK, MVD, PMD, idi, and ggps.
  • the operon comprises a heterologous promoter 5’ to the polynucleotides encoding the polypeptides. In one such embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotide CycA07.
  • heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, or 25. In one embodiment, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10.
  • the operon comprises a heterologous insulator 5’ to the polynucleotide sequence encoding CycA07.
  • the heterologous insulator is 3’ to the heterologous promoter. Any heterologous insulator may be used as appropriate for an intended purpose.
  • the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23.
  • the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11.
  • the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 10 of the genes encoding the polypeptides. Any heterologous RBS may be used as appropriate for an intended purpose.
  • the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
  • the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17, as shown below.
  • SUBSTITUTE SHEET (RULE 26) 5461 atattgatca aatcgataat gcgaaagcag ctatcttaca taaaacgaaa aaattgatag
  • the mosquito-repelling terpene comprises kolavelool.
  • the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS); TR51 15710 cyclase (CycAOl); and terpene synthase family (CycBOl).
  • a list of the genes and encoded proteins are shown in Tables 6-7, and further
  • the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of SEQ ID NO:2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO: 4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO:5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta-isomerase (IDI) comprising the amino acid sequence of SEQ ID NOT, geranylgeranyl pyrophosphate synthase (GGPS) comprising
  • the polynucleotides may be codon-optimized for expression in the bacterial cell of interest.
  • the bacterial cell may be transfected with separate polynucleotides encoding each polypeptide, with a single polynucleotide encoding all of the polypeptides, or 2 or more polynucleotides each encoding 1 one or more of the polypeptides.
  • the polynucleotides are arranged in a single operon, with all polypeptides encoded by a single polynucleotide.
  • the poly nucleotides encoding CycAOl and CycBOl are 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity.
  • the operon comprises, in 5’ to 3’ order, polynucleotides encoding CycAOl, CycBOl, atoB, HMGS, HMGR, MK, PMK, MVD, idi, and ggps.
  • the operon comprises a heterologous promoter 5’ to the polynucleotides encoding the polypeptides. In one such embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotide CycAOl. Any heterologous promoter may be used as appropriate for an intended purpose. In various embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, or 25. In one embodiment, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO:10.
  • the operon comprises a heterologous insulator 5’ to the polynucleotide sequence encoding CycAOl.
  • the heterologous insulator 5 to the polynucleotide sequence encoding CycAOl.
  • SUBSTITUTE SHEET (RULE 26) insulator is 3’ to the heterologous promoter. Any heterologous insulator may be used as appropriate for an intended purpose.
  • the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23.
  • the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11.
  • the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 10 of the genes encoding the polypeptides. Any heterologous RBS may be used as appropriate for an intended purpose.
  • the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
  • the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO:20, as shown below.
  • SUBSTITUTE SHEET 1501 cgagtcctga agaaacagct tatgctgtac tagcgcttga tttatttgca agtagaggtg
  • the disclosure provides polynucleotides comprising the nucleotide sequence of any one of SEQ ID NO: 13, 17, and 20.
  • the disclosure provides recombinant expression vectors comprising the polynucleotide "Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product.
  • "Control sequences" operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence is still considered “operably linked" to the coding sequence.
  • Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, etc.
  • Such expression vectors can be of any type known in the art
  • the present disclosure provides cells comprising the polynucleotide of the disclosure, such as a prokaryotic cell.
  • compositions comprising a plurality of the engineered bacterial cell of any previous claim and a pharmaceutically acceptable carrier.
  • the compositions may be of any suitable format for application of the compositions to the skin of a subject, including but not limited to a human subject.
  • the composition is formulated for topical application.
  • the topical formulation may be in the form of a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
  • the disclosure also provides methods to repel mosquitos, comprising contacting the skin of a subject with a plurality of the engineered bacterial cells of any embodiment or combination of embodiments herein, or with the composition of any embodiment or combination of embodiments herein, thereby repelling mosquitos from skin of the subject.
  • the disclosure provides methods to repel mosquitos, comprising contacting the skin of a subject with kaur- 16-ene, kolavelool, or a combination thereof, thereby repelling mosquitos from skin of the subject.
  • the subject may be any subject that can benefit from repelling mosquitos, including but not limited to human subjects.
  • the compositions or compounds may be administered in any suitable formulation, including being formulated for topical application.
  • the topical formulation may be in the form of a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
  • Mosquitoes are a major source of vector-bome diseases including malaria, dengue, West Nile, zika and yellow fever, affecting millions every year.
  • Vector-bome diseases account for 17% of all infectious diseases, resulting in >700,000 deaths annually (World Health Organization, 2022).
  • Minimizing contact of mosquitoes is key to preventing the transmission of illnesses.
  • Terpenes are a structurally diverse class of natural products comprising of over 70,000 compounds. Terpenes are produced by plants, fungi and bacteria, act as pheromones and are involved in a number of important ecological functions such as plant cell signaling and defense.
  • To screen for terpenes that are mosquito repellents we first used a mosquito olfaction apparatus that can quantify the repellency of terpenes that are generated by Escherichia coll strains heterologously expressing a number of different terpene pathways (Figure 1).
  • the olfactometer used here is a dual-choice apparatus for screening the behavior of molecules that contribute to mosquito repulsion (Figure 2A).
  • This behavioral assay identified two terpenes as mosquito repellents: the monoterpene eucalyptol (also known as 1,8 cineole) and a diterpene repellent kaur-16-ene (Figure 2B).
  • SUBSTITUTE SHEET (RULE 26) isolate, Staphylococcus epidermidis LM088. Manipulation of skin commensals most commonly found on humans has been particularly challenging, with only a handful of reported examples to date. These reported examples often involve introducing a single gene by plasmid transformation, which is not suitable for expressing & maintaining the multi-gene biosynthetic pathways required for terpene production. To address this challenge, we used a conjugation strategy optimized in the Voigt lab to integrate exogenous DNA in undomesticated bacteria (Voigt, Nat. Microbiol. 2018, 3, 9).
  • mini-ICE integrated-conjugative elements
  • the mosquito olfactometer was then used to assess the efficacy of these engineered, terpene producing S. epidermidis strains as mosquito repellents.
  • In vitro analysis of these engineered strains in an olfactometer showed that the eucalyptol-producing sMIH197 readily repels mosquitoes (Figure 3C). This is the first demonstration of an engineered skin microbe successfully repelling mosquitoes.
  • manipulating the skin microbiome to produce mosquito repellent terpenes such as eucalyptol and kaur-16-ene offers a novel strategy of repelling mosquitoes and minimizing transmission of mosquito associated illnesses.
  • Donor B. subtilis strains were generated by natural transformation of integrative plasmids. A single colony of B. subtilis was inoculated in 2ml of LB + 100 pg ml 1 D-alanine
  • subtilis at OD600 1, 1000 ng of plasmid DNA (containing the full terpene pathway subcloned in to pJAB980), 50 pg ml 1 tryptophan, 50 pg ml 1 phenylalanine, and 100 pg ml 1 D-alanine.
  • Cells were grown for 24 h at 37 °C and 250 r.p.m and were plated onto LB selective medium containing 100 pg ml 1 D-alanine and 5 pg ml 1 of tetracycline.
  • Successful B. subtilis colony donor strains were screened by PCR and used for conjugating primary skin commensals.
  • Donor (B. subtlis + terpene pathway) and recipient (S', epidermidis LM088) strains were picked from solid medium and inoculated in 2 mL of medium in 15 ml culture tube for 12 h at 37°C, 250 r.p.m. The dense overnight cultures were diluted 100-fold into 25 mL of medium in 250 mL Erlenmeyer flasks and grown at 37°C, 250 r.p.m. The recipient S. epidermidis LM088 strain was grown to an OD600 of 1. The donor B.
  • TSS Tris-Spizzizen Salts

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Abstract

Engineered bacterial cells for producing a mosquito-repelling terpene are provided, that include an engineered terpene biosynthetic pathway introduced into a bacterial cell that is not otherwise capable of producing the terpene, such that the engineered bacterial cell is capable of producing the mosquito-repelling terpene, and methods for using such cells to repel mosquitos.

Description

Engineering human skin microbes to produce mosquito repellent terpenes
Federal Funding Statement
This invention was made with government support under HR0011-20-2-0030 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.
Sequence Listing Statement
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on March 11, 2024 having the file name “23- 0790-WO.xml” and is 61,465 bytes in size.
Background
Mosquitoes are a major source of vector-bome diseases including malaria, dengue, West Nile, zika and yellow fever, affecting millions every year. Vector-bome diseases account for 17% of all infectious diseases, resulting in >700,000 deaths annually (World Health Organization, 2022). Minimizing contact of mosquitoes is key to preventing the transmission of illnesses.
Summary
In one aspect, the disclosure provides engineered bacterial cells for producing a mosquito-repelling terpene, comprising an engineered terpene biosynthetic pathway introduced into a bacterial cell that is not otherwise capable of producing the terpene, wherein the engineered bacterial cell is capable of producing the mosquito-repelling terpene. In one embodiment the bacterial cell is a human skin microbe; in other embodiments, the bacterial cell is Corynebacterium amycolatum, Staphylococcus caprae. or Staphylococcus epidermidis.
In one embodiment, the mosquito-repelling terpene is selected from the group consisting of eucalyptol, kaur-16-ene, and kolavelool. In one embodiment, the mosquitorepelling terpene comprises eucalyptol. In one such embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic
1
SUBSTITUTE SHEET (RULE 26) activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranyldiphosphate synthase (GPPS), and 1,8 cineole synthase (bCinS).
In another embodiment, the mosquito-repelling terpene comprises kaur- 16-ene. In one such embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: wherein the bacterial cell is transformed with polynucleotides encoding the folio-wing polypeptides having enzymatic activity: acetyl- CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS), PtmT2 cyclase (CycA07), and Hypothetical cyclase (CycB04)
In a further embodiment, the mosquito-repelling terpene comprises kolavelool. In one such embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS); TR51 15710 cyclase (CycAOl); and terpene synthase family (CycBOl).
In another aspect, the disclosure provides polynucleotides comprising the nucleotide sequence of any one of SEQ ID NO: 13, 17, and 20, recombinant expression vector comprising the polynucleotides, and recombinant host cells compnsing the polynucleotide or recombinant expression vector.
In another embodiment, the disclosure provides compositions comprising a plurality of the engineered bacterial cell of any embodiment or combination of embodiments herein, and a pharmaceutically acceptable carrier. In one embodiment, the compositions are formulated for topical administration, including but not limited to a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
In a further aspect, the disclosure provides methods to repel mosquitos, comprising contacting the skin of a subject with a plurality of the engineered bacterial cells of any preceding claim, or with the composition of any embodiment herein, thereby repelling
2
SUBSTITUTE SHEET (RULE 26) mosquitos from skin of the subject. In another aspect, the disclosure provides methods to repel mosquitos, comprising contacting the skin of a subject with kaur- 16-ene, kolavelool, or a combination thereof, thereby repelling mosquitos from skin of the subject.
Description of the Figures
Figure 1. Terpene production pipeline to produce potential mosquito repellents. (A) Outline of E. coli strain harboring biosynthetic pathway including the mevalonate pathway, a prenyl transferase and terpene cyclase genes for in vivo terpene production. This strains are cultivated in screw cap vials and analyzed by GC-MS for terpene production. B: Select list of terpenes produced in E. coli.
Figure 2. Mosquito olfactometer set up to determine repellent terpenes (A) Overview of mosquito olfactometer. Mosquitoes are placed in the start canister prior to the initiation of the experiment. A petri dish with 1 mL of bacterial culture (after cultivation for 24 h) is placed in the stimulus box. Repellency is determined by the number of mosquitoes travelling from the start canister, through the flying tube and ultimately residing in the trap after 5 minutes. (B) E. coli strains producing eucalyptol and kaur- 16-ene repel mosquitoes (box) when compared to an E. coli control. LA (lactic acid) + ammonia are used to stimulate mosquito response in the olfactometer.
Figure 3. Engineering of a primary skin isolate to repel mosquitoes. (A) Engineered S. epidermidis LM088 (sMIH197) to contain a constitutively expressed biosynthetic pathway of eucalyptol on the genome. (B) Engineered strain successfully produces eucalyptol, as detected by GCMS. (C) SMIH197 repels mosquitoes when analyzed in mosquito olfactometer described in Figure 2A.
Figure 4. Engineenng of a skin isolate to produce repellent kaur-16-ene. (A) Biosynthesis of kaur- 16-ene utilizes two diterpene cyclases (cycA07, cycB04). Successfully engineered S. epidermidis LM088 (sMI210) to constitutively express kaur-16-ene pathway. (B) Engineered strain successfully produces kaur- 16-ene, as detected by GCMS.
Figure 5. Engineering of a primary' skin isolate produce Kolavelool. Biosynthesis of kolavelool utilizes two diterpene cyclases (cycAOl, cycBOl). Successfully engineered A epidermidis LM088 (sMI217) to constitutively express kolavelool pathway. (B) Engineered strain successfully produces kolavelool, as detected by GCMS
Figure 6. Olfactometer analysis of S. epidermidis eucalyptol producer (S. epi Euc.), S. epidermidis kaur-16-ene producer (S. epi Kaur.) and S. epidermidis kolavelool producer
3
SUBSTITUTE SHEET (RULE 26) (S. epi Koi.). Positive control: Lactic acid + Ammonia (attractant). Negative control: 2- methylbutyric acid (repellent).
Detailed Description
As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘compnse’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the words “herein,” “above” and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
As used herein, "about" will mean up to plus or minus 5% of the particular value.
In one aspect, the disclosure provides engineered bacterial cells for producing a mosquito-repelling terpene, comprising an engineered terpene biosynthetic pathway introduced into a bacterial cell that is not otherwise capable of producing the terpene, wherein the engineered bacterial cell is capable of producing the mosquito-repelling terpene.
As shown in the examples that follow, the inventors have identified mosquito- repellant terpenes and heterologously expressed terpene biosynthetic pathways in human skin microbes that can successfully generate the mosquito-repellant terpenes.
As used herein, “mosquito-repellant terpene” means a terpene that deters mosquitoes from approaching or settling on a subject.
The engineered bacterial cells may be any bacteria that is not otherwise capable of producing the mosquito-repellant terpene. In one embodiment, the bacterial cells are human skin microbes. Many such human skin microbes are known and included in the present disclosure. In some embodiments, the bacterial cell may be a primary isolate from human skin. In other embodiments, the bacterial cell may be one that is available from a depository or commercially. In certain embodiments, the bacterial cells comprise Corynebacterium amycolatum, Staphylococcus caprac. or Staphylococcus epidermidis. Exemplary sources for such cells include, but are not limited to, primary isolates isolated using standard techniques
4
SUBSTITUTE SHEET (RULE 26) (including but not limited to Staphylococcus epidermidis LM088), Corynebacterium amycolatum ATCC 49368, Staphylococcus caprae ATCC 55133, and Staphylococcus epidermidis ATCC 12228.
In certain embodiments, the mosquito-repelling terpene is selected from the group consisting of eucalyptol, kaur-16-ene, and kolavelool. The structures of these compounds are shown below.
E
Figure imgf000006_0001
In one embodiment, the mosquito-repelling terpene comprises eucalyptol. In this embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranyldiphosphate synthase (GPPS), and 1,8 cineole synthase (bCinS). A list of the genes and encoded proteins are shown in Tables 1-2, and further details are provided in the examples.
Table 1: List of genes for the biosynthesis of Eucalyptol
Figure imgf000006_0002
5
SUBSTITUTE SHEET (RULE 26)
Figure imgf000007_0001
In one embodiment, the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of
SEQ ID NO: 2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO: 5, diphosphomevalonate decarboxylase (MVD) comprising the ammo acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta-isomerase (IDI) comprising the amino acid sequence of SEQ ID NO:7, geranyldiphosphate synthase (GPPS) comprising the amino acid sequence of SEQ ID NO:8, and 1,8 cineole synthase (bCinS) comprising the amino acid sequence of SEQ ID NO:9. Table 2. Amino acid sequence of encoded proteins
Figure imgf000007_0002
SUBSTITUTE SHEET (RULE 26)
Figure imgf000008_0001
7
SUBSTITUTE SHEET (RULE 26)
Figure imgf000009_0001
In all embodiments disclosed herein, the polynucleotides may be codon-optimized for expression in the bacterial cell of interest.
8
SUBSTITUTE SHEET (RULE 26) In all embodiments disclosed herein, the bacterial cell may be transfected with separate polynucleotides encoding each polypeptide, with a single polynucleotide encoding all of the polypeptides, or 2 or more polynucleotides each encoding 1 one or more of the polypeptides. In one embodiment, the polynucleotides are arranged in a single operon, with all polypeptides encoded by a single polynucleotide. The polynucleotides encoding the different polypeptides may be in any order in the operon. In one embodiment, the polynucleotide encoding bCinS is 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity. In a further embodiment, the operon comprises, in 5’ to 3’ order, polynucleotides encoding bCmS, atoB, HMGS, HMGR,MK, PMK, MVD, idi, and gpps.
In one embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotides encoding the polypeptides. In one such embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotide bCinS.
As used throughout herein, a “heterologous” promoter, insulator, or ribosome binding site is one that not naturally occurring as a regulatory' sequence for the genes it is controlling expression of. Any heterologous promoter may be used as appropriate for an intended purpose. In various embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NOTO, 21, 22, 24, or 25. In one embodiment, the heterologous promoter comprises the nucleotide sequence of SEQ ID NOTO.
In another embodiment, the operon comprises a heterologous insulator 5’ to the polynucleotide sequence encoding bCinS. In a further embodiment, the heterologous insulator is 3’ to the heterologous promoter. Any heterologous insulator may be used as appropriate for an intended purpose. In some embodiments, the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23. In one embodiment, the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11.
In a further embodiment, the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 9 of the genes encoding the polypeptides. Any heterologous RBS may be used as appropriate for an intended purpose. In one embodiment, the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
Table 3. Sequences of regulatory elements
Figure imgf000010_0001
SUBSTITUTE SHEET (RULE 26)
Figure imgf000011_0001
In a specific embodiment, the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13, as shown below.
SEQ ID NO : 13
LOCUS Eucalyptol_Pathw 10078 bp DNA
1 cggtggaaac gaggtcatca tttccttccg aaaaaacggt tgcatttaaa tcttacatat
61 gtaatacttt caaagactac atttgtaaga tttgaagctg tcaccggatg tgctttccgg
121 tctgatgagt ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa ttaggaggat
181 gattatttat gcctgcaggt catgaagaat tcgatatacc atttccttca cgtgtaaatc
241 catttcatgc acgagcagaa gatcgtcatg ttgcttggat gagagctatg ggtttaatta
301 cgggtgatgc tgctgaagcg acttatcgtc gttggtcacc ggctaaagta ggtgcaagat
361 ggttttatct tgctcaaggt gaagatttag atttgggttg tgacattttt ggatggtttt
421 tcgcttatga tgaccatttt gatggtccga ctggcactga tccaagacaa actgcagctt
481 ttgttaatag aactgtcgca atgttagatc ctcgtgcaga tcctacaggt gaacatccat
541 taaatattgc atttcatgat ctatggcaaa gagagagtgc acctatgagt ccattatggc
601 aacgacgtgc tgttgatcac tggacacaat atttaacagc tcatattact gaagcaacta
661 atcgtacaag acatacatca cctacaattg cagattactt agaattacga catagaactg
721 gatttatgcc gccattgctt gacttaattg aaagagtatg gcgtgctgaa atacctgcgc
781 ctgtatatac gacaccagag gtgcaaacat tgctacacac aacaaatcag aatatcaaca
SUBSTITUTE SHEET (RULE 26) 841 tagttaatga cgttttatct cttgagaaag aggaagcgca cggcgatcca cataacctag
901 tattagtgat tcaacatgaa agacaaagta ctagacaaca agcattagca actgcacgaa
961 gaatgattga tgaatggact gatacattca tccgtactga accaagactt ccagcattat
1021 gcggacgatt aggaatacca cttgctgatc gtacatcttt atacacagct gtcgaaggta
1081 tgcgtgctgc aattagagga aattatgatt ggtgtgctga aacaaatcgt tatgctgttc
1141 atcgtcctac aggaacgggt cgtgcgacga ctccttggta attaggagga tgattattta
1201 tggctgaagc ttatattgtt gaagcagtta gaacgcctgt cggtagaaga cgaggtggat
1261 tagctggcgt acacccagct gatttaggag cacatgcttt aacagctcta gttgcacgtt
1321 caggaattga tcctgcagcg gttgaagatg ttgtatttgg ttgcttagat acagttggtc
1381 cacaggctgg tgatattgca agaacttgtt ggttagcagc aggtcttcct gaagaagtac
1441 caggagtgac agtagataga caatgtggaa gttcacaaca agctgtgcat tttgctgcac
1501 aagctgttct atcaggtact caggatttgg tggtcgctgg aggagttcaa aatatgtcac
1561 aaattccaat agcatttgct tctagacaag cagctgaacc tttaggttta acgcaaggac
1621 cattcgcagg ttctgaaggt tggcgtgctc gatatggtga tcttccagtt aatcaatttc
1681 atggcgcaga actaattgcg gcaaagtggg gaattacacg acgtgatcaa gaagaatttg
1741 cattaagatc acatagaaga gcggtacgtg catctgacga aggtagattt gaccgagaaa
1801 cggttgcgta tggtaaagtc actgcggatg agggaccacg tagagataca agtttagaaa
1861 aaatggcggc tttagcacca gtaattgaag gtggaacgat tacagcagca tgttcaagtc
1921 aagtaagtga tggcgcagct gctatgttat tggcttcaga acgtgctttg agagaacatg
1981 gattaacgcc tagagcacgt gtgcatcatc taagtgtaag aggagaagat ccaattagaa
2041 tgttatctgc acctatccct gcaacagcac acgcattaaa aaaaactgct ttagcaattg
2101 atgatataga tttagtagaa atcaatgaag cgttcgcacc tgttgtactt gcatggttaa
2161 aagagactgg agcagatcct gaaaaagtga atgtcaatgg tggcgcaatc gctttaggac
2221 atcctttagg tgctacaggt gttagattga tgacgacatt attacacgag ttagaaagaa
2281 ctggtggtcg ttttggttta caaacaatgt gtgaaggagg aggccaggcg aatgttacga
2341 tcatagaaag actttagtta ggaggatgat tatttatgac tataggtata gacaaaatag
2401 gtttcgctac atctcaatat gttttaaaac tagaagattt agcattggca cgtcaagtag
2461 atccagcaaa gttttctcaa ggtttattaa ttgaatcatt ttctgtagca cctatcacag
2521 aagatattat tacgttagca gcttctgcag cggaccaaat cctaactgat gaagatcgtg
2581 caaaaataga catggtaata ttagcaactg aaagtagtac tgaccaatca aaagcaagtg
2641 ctatttatgt tcaccatctt gtgggcattc aaccatttgc acgaagtttt gaagtaaagc
2701 aagcttgcta cagtgctaca gcagcattag attatgcaaa attacatgta gcaagtaaac
2761 cagatagtag agtattagtg atagcttcag acattgctcg ttatggagta ggatcaccag
2821 gagaaagtac acaaggaagt ggtagtattg cattgttggt gacagcaaat ccacgtatct
2881 tagcattaaa taaagataat gttgctcaaa cacgtgatat aatggatttc tggcgaccaa
2941 attactcatt tacaccatac gttgatggta tttatagtac taaacaatat cttaattgct
3001 tagagactac atggcaagct tatcagaaac gagaaaattt acaattaagt gattttgctg
3061 cagtctgttt tcatatccct tttcctaaat tagcattaaa gggtttaaat aacattatgg
3121 ataatactgt ccctccagag catagagaaa aactaattga agcgttccaa gcatcaatta
3181 cttattctaa acaaattggc aatatttata caggaagtct ttacttagga ttattgagtc
3241 ttttagaaaa tagtaaagtt ttacagtctg gtgacaagat cggttttttc tcatatggct
11
SUBSTITUTE SHEET (RULE 26) 3301 caggagcagt ctctgaattt tattcaggcc aacttgttgc aggttatgat aaaatgttaa
3361 tgacaaatcg acaagcactt ttggatcaaa gaactagatt gtcagtttct aaatacgaag
3421 atttatttta tgaacaagtt caacttgatg ataatggtaa tgctaatttt gatatatatc
3481 taacaggtaa atttgctctt acagcaatca aagagcacca aagaatttac cacactaatg
3541 ataaaaatta attaggagga tgattattta tgactaaaac gaatttaaat tggtctggtt
3601 tctctaaaaa aacatttgaa gagcgtttac aattaataga aaaatttaaa ttactaaatg
3661 cagaaaactt aaatcaatta aaaacagatg ttttattacc tattcaaacg gcgaaccaga
3721 tgacagagaa tgtgttaggt cgtttagcat tacctttttc aatagcgcca gactttttag
3781 ttaatggttc tacataccaa atgccatttg tcacagaaga gccttctgtg gtagctgcag
3841 caagttttgc tgcaaagtta ataaaaagaa gtggtggatt taaagcgcaa acacttaatc
3901 gtcaaatgat cggccaaatc gtgttatacg atattgatca aatcgataat gcgaaagcag
3961 ctatcttaca taaaacgaaa aaattgatag ctttggctaa caaagcatat ccatcaattg
4021 ttaaacgagg tggtggcgca agaactattc atttggaaga gaaaggtgaa ttcttaatat
4081 tttatttaac ggtagacaca caagaagcaa tgggtgctaa tatggtcaat acaatgatgg
4141 aagctttggt tccagactta actcgtttga gtaagggaca ttgtttaatg gcaatattat
4201 caaattatgc tacagaatca ctagttacaa catcttgcga aataccagtt agattattag
4261 atcgtgacaa aacaaaatct ttacaattag cacaaaaaat tgaattagca agtcgtctag
4321 ctcaagtgga tccttatcgt gcaacaacac ataataaagg tatttttaat ggtatagacg
4381 ctgtcgtcat tgctactggt aacgactggc gtgcaattga agcaggtgca catgcatatg
4441 cttcacgaaa tggtagttat caaggcttaa gtcaatggca ttttgatcaa gacaaacagg
4501 ttttattagg tcaaatgaca ttacctatgc caattgcttc aaaaggaggt tcaataggct
4561 taaatcctac tgttagtatt gcacatgatt tattaaatca accagatgct aaaacactag
4621 ctcaacttat tgcaagtgta ggtttagcac aaaactttgc agcactaaaa gcgttaacta
4681 gttcaggtat ccaagcaggt catatgaaat tacacgcgaa atctcttgct ttattggcag
4741 gcgctactca agatgaaatt gcaccattgg taaacgcttt gcttgctgat aaaccaatta
4801 atctagaaaa agctcacttt tatttatcac aactacgtca aagttgagtc gacctgcagg
4861 catgcaagct tggctgtttt ggttaggagg atgattattt atgaacgaga atataggtta
4921 tggaaaagca cattctaaaa ttattttaat aggtgaacat gcagtagtat atggctatcc
4981 agcaattgct cttcctctta cagatataga agtagtgtgt catatttttc cagcggataa
5041 accactagtt tttgacttct atgacacttt aagtacagca atatatgcgg cacttgatta
5101 tcttcaaaga ttgcaagaac caatagctta cgaaatagtt agtcaagtac cacagaagcg
5161 tggcatgggt agtagtgctg ctgttagtat tgcagcgatt agagctgttt tttcttattg
5221 tcaagagcct ttaagtgacg atttacttga aatcctagta aataaagcag aaataattgc
5281 acacacaaat ccttcaggat tagatgctaa aacatgctta tctgatcacg ctataaaatt
5341 tattagaaat ataggttttg agacaatcga aattgcttta aatggttatc taattattgc
5401 tgatacgggt attcatggtc atacaagaga agctgttaat aaagtcgctc agtttgaaga
5461 aacaaattta ccatatttag caaaacttgg tgcattaaca caagcattag aaagagctat
5521 caaccaaaaa aataaggtag caattggaca attaatgaca caagctcata gtgcattgaa
5581 agcaattggt gtatctatct ctaaagctga tcaattagtc gaagcagcat tacgagcagg
5641 tgcattaggt gctaaaatga caggtggtgg tctaggtggt tgtatgattg ctttagcaga
5701 tacaaaagat atggcagaaa aaatttcaca tagattaaag gaggaaggag cggtaaatac
12
SUBSTITUTE SHEET (RULE 26) 5761 atggattcag atgttatagt taggaggatg attatttatg agtaactact gtgtacaaac
5821 gggcggtaaa ttatatttaa caggagaata tgcaatactt attccaggtc aaaaagcttt
5881 aattcacttt attccattaa tgatgactgc tgaaatcagt ccagctgcac atattcaact
5941 tgcttcagat atgttttcac acaaagcggg tatgacacct gatgcaagtt atgctttaat
6001 tcaagcaaca gttaaaacat ttgcagatta cctaggacaa agtattgatc aattagaacc
6061 tttcagtctt attatcacgg gtaaaatgga acgtgatgga aaaaaattcg gtataggaag
6121 ttcaggatct gttactttat taactttaaa agcattgtca gcttattatc aaatcacatt
6181 aacaccagaa ttgttattta aattagctgc atatactttg ctaaaacagg gagataacgg
6241 ttctatggga gacatagcgt gcattgctta ccaaacatta gttgcatata catcttttga
6301 tcgtgaacaa gtgtcaaatt ggttacaaac tatgccatta aaaaaacttt tagtcaaaga
6361 ttggggatat catattcaag taattcagcc tgctttgcct tgtgatttct tagttggttg
6421 gactaagata cctgcaattt cacgtcaaat gattcagcaa gtaacggctt ctattacacc
6481 tgcgttttta cgtacgtcat atcaattaac acaatctgct atggtggctt tacaagaagg
6541 ccataaagaa gaattaaaaa aatcattggc tggagcttca cacttattaa aagaattaca
6601 ccctgctatt tatcatccta aattggttac attagttgct gcttgtcaaa aacaggatgc
6661 agtagcaaaa agtagtggaa gtggtggagg cgactgcggt attgctcttg catttaatca
6721 agatgcgcgt gatacattaa tttcaaaatg gcaagaagct gatatagctc ttttatacca
6781 agaaagatgg ggagagaatg actagttagg aggatgatta tttatggatc ctaatgtaat
6841 cacggttact tcatatgcaa atatagcaat aattaaatac tggggtaaag aaaatcaagc
6901 taaaatgatc ccttctacat cttctatttc attgacatta gaaaatatgt tcactactac
6961 ttcagtgtca tttttaccag acactgcaac ttcagatcaa ttttatataa acggtgtgtt
7021 acaaaatgat gaagaacata cgaaaatatc aacaattatt gatcaattcc gtcaaccagg
7081 acaagctttt gtaaaaatgg aaactcaaaa taacatgcct acagctgcag gattatcatc
7141 atcatcttca ggtttgtctg cattagtaaa ggcttgtgat caattgtttg atacacagct
7201 agatcaaaaa gcattagcac aaaaagcaaa atttgcgtct ggaagttcat cacgttcttt
7261 ctttggtcct gtcgcggcat gggataaaga ttctggcgca atatataaag ttgaaacgga
7321 cttgaaaatg gcgatgatta tgttagtttt aaacgcagca aaaaaaccta tttcatcacg
7381 tgaaggtatg aaattatgtc gtgatacatc aacaacattt gatgaatggg ttgaacagtc
7441 agcaattgac taccaacata tgttgacata tttaaaaaca aacaacttcg aaaaagttgg
7501 ccaattaaca gaagcgaatg cattggctat gcatgcaact acaaagacag cgaatcctcc
7561 ttttagttac ctaactaaag aatcttatca agctatggaa gctgtaaaag aattgcgtca
7621 agaaggtttt gcatgctatt ttacaatgga tgcaggacct aatgtcaaag tactttgttt
7681 agagaaagat ttagcacaat tggcagagag attgggaaaa aattatcgaa ttatagtatc
7741 aaaaacaaaa gacttgccag acgtttaatt aggaggatga ttatttatga ctaatagaaa
7801 agatgatcat attaagtatg ctcttaaata ccaatcacct tataacgctt ttgatgatat
7861 tgaattaatt catcatagtc taccttctta tgatcttagt gacattgatt tatcaactca
7921 ctttgcagga caagattttg actttccatt ttatattaac gctatgacag gaggatctca
7981 aaaaggtaaa gctgttaatg aaaaattagc aaaagttgca gctgcaacag gtattgtaat
8041 ggtaacagga tcttatagtg cagctttgaa aaatccaaat gatgattctt atagattaca
8101 cgaggttgct gacaatctta aattagctac taacattggt ttagacaagc ctgttgcttt
8161 aggtcagcag acagtgcaag aaatgcaacc tttgtttctt caagtacatg ttaacgttat
13
SUBSTITUTE SHEET (RULE 26) 8221 gcaagaattg ttaatgccag aaggtgaaag agtttttcat acttggaaaa aacatctagc
8281 tgaatatgct agtcaaatac cagtcccagt aattcttaaa gaagtaggct ttggtatgga
8341 tgttaactct attaaattag cacatgattt gggtattcaa actttcgata taagtggtag
8401 aggtggaact agtttcgctt atatagaaaa tcaacgtggt ggcgatagat catacttgaa
8461 tgactggggt caaactacag ttcagtgtct tttaaatgca caaggcctaa tggatcaagt
8521 cgaaatatta gcatcaggtg gtgtacgtca tcctcttgat atgattaaat gttttgtttt
8581 aggcgctcgt gcagtaggat tatctagaac ggtgttggaa ttagttgaaa aatatccaac
8641 tgagagagta atcgctatag taaatggttg gaaggaagaa ttgaaaataa ttatgtgtgc
8701 attggattgc aaaacaataa aagagctaaa aggcgtggat tatttattat acggacgact
8761 tcaacaagta aattaagcga agacttaatc attaggagga tgattattta tggcttattc
8821 agcaatggct actatgggtt ataacggtat ggctgcatca tgtcatacac ttcatccaac
8881 atctccatta aaaccttttc acggtgcatc aacttcactt gaagcattta atggagaaca
8941 tatgggttta cttcgtggat atagtaaacg aaaattatct agttataaaa atcctgcatc
9001 tagatcatca aatgcaacgg ttgcacaatt attaaatcca ccacaaaaag gaaagaaagc
9061 tgtagaattt gattttaata aatatatgga tagtaaagct atgacagtta atgaagcgtt
9121 aaataaggct attcctctaa gatatccaca aaaaatttat gaaagtatgc gttattcatt
9181 attagcaggt ggtaaacgag tacgtcctgt cttatgcata gcagcttgtg aattagttgg
9241 tggtactgaa gaactagcta ttccaacagc gtgtgctatc gaaatgattc acactatgtc
9301 attgatgcat gatgatttac catgtattga taatgatgat ttaagaagag gtaaaccaac
9361 aaatcataaa atctttggtg aagacacagc tgttacagca ggcaacgcgt tacattcata
9421 tgcattcgag catatagctg ttagtactag taagactgtc ggagcggata gaatattacg
9481 tatggtatca gaattaggaa gagctactgg ttcagaaggt gttatgggag gtcaaatggt
9541 agatattgcg tcagaaggtg atccatcaat cgatttacaa acactagagt ggattcacat
9601 tcataaaaca gcgatgttgt tagaatgttc agtagtttgt ggagcgatta taggtggtgc
9661 aagtgaaatc gtaattgaaa gagcgcgtcg ttacgcaaga tgtgttggct tattatttca
9721 agttgttgat gatattttag atgtaacaaa atctagtgat gaattaggta aaacagcagg
9781 taaagactta atatctgata aagcaacata cccaaaattg atgggattgg agaaagcaaa
9841 agaattctct gatgaattgc ttaatagagc aaaaggtgaa ttatcatgtt ttgatccagt
9901 taaagcagct ccacttcttg gattagcaga ttatgtagca ttcagacaaa atcggatgta
9961 aacagagaaa cgtcacactg gctcaccttc gggtgggcct ttctgcgttt atagcctaat
10021 acacctgttc gtggagaata taaaaagcca gattattaat ccggcttttt tattattt
In another embodiment, the mosquito-repelling terpene comprises kaur- 16-ene. In this embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: wherein the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl -Co A C -acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD),
14
SUBSTITUTE SHEET (RULE 26) Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS), PtmT2 cyclase (CycA07), and Hypothetical cyclase (CycB04). A list of the genes and encoded proteins are shown in Tables 4-5, and further details are provided in the examples.
Table 4. List of genes for the biosynthesis of Kaur-16-ene in S. epidermidis
Figure imgf000016_0001
In one embodiment, the bactenal cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO:1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of
SEQ ID NO: 2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO: 5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta-isomerase (IDI) comprising the amino acid sequence of SEQ ID NOT; geranylgeranyl pyrophosphate synthase (GGPS) comprising the amino acid sequence of SEQ ID NO: 14; PtmT2 cyclase (CycA07) comprising the amino acid sequence of SEQ ID NO: 15; and Hypothetical cyclase (CycB04) comprising the amino acid sequence of SEQ ID NO: 16.
15
SUBSTITUTE SHEET (RULE 26) Table 5. Amino acid sequence of encoded proteins
Figure imgf000017_0001
the bacterial cell of interest. In other embodiments, the bactenal cell may be transfected with separate polynucleotides encoding each polypeptide, with a single polynucleotide encoding all of the polypeptides, or 2 or more polynucleotides each encoding 1 one or more of the polypeptides. In one embodiment, the polynucleotides are arranged in a single operon, with all polypeptides encoded by a single polynucleotide.
SUBSTITUTE SHEET (RULE 26) In one such embodiment, the polynucleotides encoding CycA07 and CycB04 are 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity. In another embodiment, the operon comprises, in 5’ to 3’ order, polynucleotides encoding CycA07, CycB04, atoB, HMGS, HMGR, MK, PMK, MVD, PMD, idi, and ggps.
In one embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotides encoding the polypeptides. In one such embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotide CycA07.
Any heterologous promoter may be used as appropriate for an intended purpose. In various embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, or 25. In one embodiment, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10.
In another embodiment, the operon comprises a heterologous insulator 5’ to the polynucleotide sequence encoding CycA07. In a further embodiment, the heterologous insulator is 3’ to the heterologous promoter. Any heterologous insulator may be used as appropriate for an intended purpose. In some embodiments, the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23. In one embodiment, the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11.
In a further embodiment, the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 10 of the genes encoding the polypeptides. Any heterologous RBS may be used as appropriate for an intended purpose. In one embodiment, the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
In another embodiment, the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17, as shown below.
SEQ ID NO: 17
LOCUS Kaur- 16-ene_Path 11551 bp DNA
1 cggtggaaac gaggtcatca tttccttccg aaaaaacggt tgcatttaaa tcttacatat 61 gtaatacttt caaagactac atttgtaaga tttgaagctg tcaccggatg tgctttccgg 121 tctgatgagt ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa ttaggaggat 181 gattatttat gctagaggta ccagctcaac ctacacctgc tccaagagag gctgaggcag 241 ccgctttgct tgccgcaaca gtagcagacc cgtggggttt ggtggccccg tctgtatatg 301 atacggcacg attagtcagt ttagctcctt ggttagacgg gcatagagag agattaggat 361 atttagtaaa ggagcaaaac caagacggta gttggggagc accagatgga tacggattag 421 taccgacact atcagcagta gaggctttat taacagagtt ggctagaaca gactcaggcg 481 caccacaccc acctcatgac gacctagcag ccgcatgtgc tggtggctta ggggctctac
17
SUBSTITUTE SHEET (RULE 26) 541 aagacggttt gttagcaggt cctgtgccag acacaattgg agtagaattc gtggcaccat
601 ctcttttagc agacattaat actagactag cagccttaac tgagcaggca ccgggtaagt
661 taggtgcttg gtcaggcact acattaactt ctcctgcacc agacttagac ggtgcgttac
721 tagcaggtgt acgagaaatg acagaacaag cacctttacc tgaaaagctt tggcacactt
781 tagaggcaat tacacgtgac ggcacaagag gcgctcgtcc acacgaggga gctccacctc 841 ataacggtag tgtgggatgc tcacctgctg caactgcggc ttggctagga gcctcaccag 901 acccagctgc acctggggtt gcttatttaa gagacgtcca agcgagattc ggtggacctg 961 tcccatctat aacacctata gtttatttcg aacaagcttg ggttttaaac tctcttgcag 1021 ctagtggttt acgttacgag gcaccagctg ctcttctaga ttcacttgag gcgggactta 1081 cagacgaggg tatagcagct gctcctggtc ttccatcaga ttcagatgac acagcggctg 1141 tcttatttgc attagcacaa catggacgaa ctcaccgtcc ggatagtcta atgcatttta 1201 gaagagacgg ttacttcagt tgcttcggag tggaacgtac tccgagtaca tctactaatg 1261 cacacatttt agaggcttta ggccatcacg tgactgttag accagatgac gcaggacgat 1321 acggagctga gattcgaatg atcagtgatt ggttactaga caaccagctt cctgacggtt 1381 catggatgga caagtggcat gctagtcctt actatgctac agcttgttgc gcattagcct 1441 tagcagagtt cggtggtcca tcagcacgag cagctgtaga cagagcagct gcatgggcgt 1501 tagagacgca gcgagcagac ggttcatggg gacgttggca aggtacgact gaggagactg 1561 cttatatggt acaactactt atgcgtacac gaactccagg atctcctgga acagtagcac 1621 gttcagccgc tagaggttgc gacgcgttac ttgcgcacga cgaccctgca tcatatcctg 1681 gtttatggca cgacaaggat atatacgcac cagtcactgt tataagagca gcgcgattgg 1741 cagctttagc gcttggtgga gctgaaagtg cggcttctgg tggagcttga ttaggaggat 1801 gattatttat gatacaaaca gaaagagcag ttcaacaagt acttgagtgg ggtagatctt 1861 taacgggctt tgctgacgag catgcggttg aagccgtgcg tggtggacaa tatatcttgc 1921 agagaataca cccaagtttg agaggaactt ctgcgcgtac tggacgtgac cctcaggacg 1981 agacattaat agtaacattt taccgtgagt tggcattact tttctggtta gatgattgca 2041 atgacttagg ccttatatca cctgagcagt tggctgctgt ggagcaagct cttggtcagg 2101 gagtcccttg tgcactacca ggtttcgagg gatgcgctgt tcttcgtgcc tcattagcaa 2161 cattggcata cgaccgtcgt gattacgcac agttattaga tgacactcgt tgctattcag 2221 cagcattacg tgctggacat gcgcaggcag ttgcagctga aagatggagt tacgcagagt 2281 atttacataa cggtattgat agtatcgctt atgcgaatgt tttctgttgc ctttcattgt 2341 tatggggatt agacatggca acattaagag cacgaccagc cttccgtcag gtgttaagac 2401 ttatttcagc gattggaaga ttacagaacg atttgcatgg atgtgataag gatcgtagtg 2461 caggagaagc tgacaacgcg gtaatattat tactacaacg ttatcctgct atgccagtgg 2521 ttgagttctt gaatgacgag cttgctggtc acacacgtat gttacataga gtaatggcag 2581 aagaaagatt ccctgctcct tggggtccat tgattgaggc aatggctgca atacgtgtcc 2641 agtactacag aactagtaca tcaagatacc gttcagacgc tgttcgaggc ggtcaacgtg 2701 cacctgctta attaggagga tgattattta tggctgaagc ttatattgtt gaagcagtta 2761 gaacgcctgt cggtagaaga cgaggtggat tagctggcgt acacccagct gatttaggag 2821 cacatgcttt aacagctcta gttgcacgtt caggaattga tcctgcagcg gttgaagatg 2881 ttgtatttgg ttgcttagat acagttggtc cacaggctgg tgatattgca agaacttgtt 2941 ggttagcagc aggtcttcct gaagaagtac caggagtgac agtagataga caatgtggaa
18
SUBSTITUTE SHEET (RULE 26) 3001 gttcacaaca agctgtgcat tttgctgcac aagctgttct atcaggtact caggatttgg
3061 tggtcgctgg aggagttcaa aatatgtcac aaattccaat agcatttgct tctagacaag
3121 cagctgaacc tttaggttta acgcaaggac cattcgcagg ttctgaaggt tggcgtgctc
3181 gatatggtga tcttccagtt aatcaatttc atggcgcaga actaattgcg gcaaagtggg
3241 gaattacacg acgtgatcaa gaagaatttg cattaagatc acatagaaga gcggtacgtg
3301 catctgacga aggtagattt gaccgagaaa cggttgcgta tggtaaagtc actgcggatg
3361 agggaccacg tagagataca agtttagaaa aaatggcggc tttagcacca gtaattgaag
3421 gtggaacgat tacagcagca tgttcaagtc aagtaagtga tggcgcagct gctatgttat
3481 tggcttcaga acgtgctttg agagaacatg gattaacgcc tagagcacgt gtgcatcatc
3541 taagtgtaag aggagaagat ccaattagaa tgttatctgc acctatccct gcaacagcac
3601 acgcattaaa aaaaactgct ttagcaattg atgatataga tttagtagaa atcaatgaag
3661 cgttcgcacc tgttgtactt gcatggttaa aagagactgg agcagatcct gaaaaagtga
3721 atgtcaatgg tggcgcaatc gctttaggac atcctttagg tgctacaggt gttagattga
3781 tgacgacatt attacacgag ttagaaagaa ctggtggtcg ttttggttta caaacaatgt
3841 gtgaaggagg aggccaggcg aatgttacga tcatagaaag actttagtta ggaggatgat
3901 tatttatgac tataggtata gacaaaatag gtttcgctac atctcaatat gttttaaaac
3961 tagaagattt agcattggca cgtcaagtag atccagcaaa gttttctcaa ggtttattaa
4021 ttgaatcatt ttctgtagca cctatcacag aagatattat tacgttagca gcttctgcag
4081 cggaccaaat cctaactgat gaagatcgtg caaaaataga catggtaata ttagcaactg
4141 aaagtagtac tgaccaatca aaagcaagtg ctatttatgt tcaccatctt gtgggcattc
4201 aaccatttgc acgaagtttt gaagtaaagc aagcttgcta cagtgctaca gcagcattag
4261 attatgcaaa attacatgta gcaagtaaac cagatagtag agtattagtg atagcttcag
4321 acattgctcg ttatggagta ggatcaccag gagaaagtac acaaggaagt ggtagtattg
4381 cattgttggt gacagcaaat ccacgtatct tagcattaaa taaagataat gttgctcaaa
4441 cacgtgatat aatggatttc tggcgaccaa attactcatt tacaccatac gttgatggta
4501 tttatagtac taaacaatat cttaattgct tagagactac atggcaagct tatcagaaac
4561 gagaaaattt acaattaagt gattttgctg cagtctgttt tcatatccct tttcctaaat
4621 tagcattaaa gggtttaaat aacattatgg ataatactgt ccctccagag catagagaaa
4681 aactaattga agcgttccaa gcatcaatta cttattctaa acaaattggc aatatttata
4741 caggaagtct ttacttagga ttattgagtc ttttagaaaa tagtaaagtt ttacagtctg
4801 gtgacaagat cggttttttc tcatatggct caggagcagt ctctgaattt tattcaggcc
4861 aacttgttgc aggttatgat aaaatgttaa tgacaaatcg acaagcactt ttggatcaaa
4921 gaactagatt gtcagtttct aaatacgaag atttatttta tgaacaagtt caacttgatg
4981 ataatggtaa tgctaatttt gatatatatc taacaggtaa atttgctctt acagcaatca
5041 aagagcacca aagaatttac cacactaatg ataaaaatta attaggagga tgattattta
5101 tgactaaaac gaatttaaat tggtctggtt tctctaaaaa aacatttgaa gagcgtttac
5161 aattaataga aaaatttaaa ttactaaatg cagaaaactt aaatcaatta aaaacagatg
5221 ttttattacc tattcaaacg gcgaaccaga tgacagagaa tgtgttaggt cgtttagcat
5281 tacctttttc aatagcgcca gactttttag ttaatggttc tacataccaa atgccatttg
5341 tcacagaaga gccttctgtg gtagctgcag caagttttgc tgcaaagtta ataaaaagaa
5401 gtggtggatt taaagcgcaa acacttaatc gtcaaatgat cggccaaatc gtgttatacg
19
SUBSTITUTE SHEET (RULE 26) 5461 atattgatca aatcgataat gcgaaagcag ctatcttaca taaaacgaaa aaattgatag
5521 ctttggctaa caaagcatat ccatcaattg ttaaacgagg tggtggcgca agaactattc
5581 atttggaaga gaaaggtgaa ttcttaatat tttatttaac ggtagacaca caagaagcaa
5641 tgggtgctaa tatggtcaat acaatgatgg aagctttggt tccagactta actcgtttga
5701 gtaagggaca ttgtttaatg gcaatattat caaattatgc tacagaatca ctagttacaa
5761 catcttgcga aataccagtt agattattag atcgtgacaa aacaaaatct ttacaattag
5821 cacaaaaaat tgaattagca agtcgtctag ctcaagtgga tccttatcgt gcaacaacac
5881 ataataaagg tatttttaat ggtatagacg ctgtcgtcat tgctactggt aacgactggc
5941 gtgcaattga agcaggtgca catgcatatg cttcacgaaa tggtagttat caaggcttaa
6001 gtcaatggca ttttgatcaa gacaaacagg ttttattagg tcaaatgaca ttacctatgc
6061 caattgcttc aaaaggaggt tcaataggct taaatcctac tgttagtatt gcacatgatt
6121 tattaaatca accagatgct aaaacactag ctcaacttat tgcaagtgta ggtttagcac
6181 aaaactttgc agcactaaaa gcgttaacta gttcaggtat ccaagcaggt catatgaaat
6241 tacacgcgaa atctcttgct ttattggcag gcgctactca agatgaaatt gcaccattgg
6301 taaacgcttt gcttgctgat aaaccaatta atctagaaaa agctcacttt tatttatcac
6361 aactacgtca aagttgagtc gacctgcagg catgcaagct tggctgtttt ggttaggagg
6421 atgattattt atgaacgaga atataggtta tggaaaagca cattctaaaa ttattttaat
6481 aggtgaacat gcagtagtat atggctatcc agcaattgct cttcctctta cagatataga
6541 agtagtgtgt catatttttc cagcggataa accactagtt tttgacttct atgacacttt
6601 aagtacagca atatatgcgg cacttgatta tcttcaaaga ttgcaagaac caatagctta
6661 cgaaatagtt agtcaagtac cacagaagcg tggcatgggt agtagtgctg ctgttagtat
6721 tgcagcgatt agagctgttt tttcttattg tcaagagcct ttaagtgacg atttacttga
6781 aatcctagta aataaagcag aaataattgc acacacaaat ccttcaggat tagatgctaa
6841 aacatgctta tctgatcacg ctataaaatt tattagaaat ataggttttg agacaatcga
6901 aattgcttta aatggttatc taattattgc tgatacgggt attcatggtc atacaagaga
6961 agctgttaat aaagtcgctc agtttgaaga aacaaattta ccatatttag caaaacttgg
7021 tgcattaaca caagcattag aaagagctat caaccaaaaa aataaggtag caattggaca
7081 attaatgaca caagctcata gtgcattgaa agcaattggt gtatctatct ctaaagctga
7141 tcaattagtc gaagcagcat tacgagcagg tgcattaggt gctaaaatga caggtggtgg
7201 tctaggtggt tgtatgattg ctttagcaga tacaaaagat atggcagaaa aaatttcaca
7261 tagattaaag gaggaaggag cggtaaatac atggattcag atgttatagt taggaggatg
7321 attatttatg agtaactact gtgtacaaac gggcggtaaa ttatatttaa caggagaata
7381 tgcaatactt attccaggtc aaaaagcttt aattcacttt attccattaa tgatgactgc
7441 tgaaatcagt ccagctgcac atattcaact tgcttcagat atgttttcac acaaagcggg
7501 tatgacacct gatgcaagtt atgctttaat tcaagcaaca gttaaaacat ttgcagatta
7561 cctaggacaa agtattgatc aattagaacc tttcagtctt attatcacgg gtaaaatgga
7621 acgtgatgga aaaaaattcg gtataggaag ttcaggatct gttactttat taactttaaa
7681 agcattgtca gcttattatc aaatcacatt aacaccagaa ttgttattta aattagctgc
7741 atatactttg ctaaaacagg gagataacgg ttctatggga gacatagcgt gcattgctta
7801 ccaaacatta gttgcatata catcttttga tcgtgaacaa gtgtcaaatt ggttacaaac
7861 tatgccatta aaaaaacttt tagtcaaaga ttggggatat catattcaag taattcagcc
20
SUBSTITUTE SHEET (RULE 26) 7921 tgctttgcct tgtgatttct tagttggttg gactaagata cctgcaattt cacgtcaaat 7981 gattcagcaa gtaacggctt ctattacacc tgcgttttta cgtacgtcat atcaattaac 8041 acaatctgct atggtggctt tacaagaagg ccataaagaa gaattaaaaa aatcattggc 8101 tggagcttca cacttattaa aagaattaca ccctgctatt tatcatccta aattggttac 8161 attagttgct gcttgtcaaa aacaggatgc agtagcaaaa agtagtggaa gtggtggagg 8221 cgactgcggt attgctcttg catttaatca agatgcgcgt gatacattaa tttcaaaatg 8281 gcaagaagct gatatagctc ttttatacca agaaagatgg ggagagaatg actagttagg 8341 aggatgatta tttatggatc ctaatgtaat cacggttact tcatatgcaa atatagcaat 8401 aattaaatac tggggtaaag aaaatcaagc taaaatgatc ccttctacat cttctatttc 8461 attgacatta gaaaatatgt tcactactac ttcagtgtca tttttaccag acactgcaac 8521 ttcagatcaa ttttatataa acggtgtgtt acaaaatgat gaagaacata cgaaaatatc 8581 aacaattatt gatcaattcc gtcaaccagg acaagctttt gtaaaaatgg aaactcaaaa 8641 taacatgcct acagctgcag gattatcatc atcatcttca ggtttgtctg cattagtaaa 8701 ggcttgtgat caattgtttg atacacagct agatcaaaaa gcattagcac aaaaagcaaa 8761 atttgcgtct ggaagttcat cacgttcttt ctttggtcct gtcgcggcat gggataaaga 8821 ttctggcgca atatataaag ttgaaacgga cttgaaaatg gcgatgatta tgttagtttt 8881 aaacgcagca aaaaaaccta tttcatcacg tgaaggtatg aaattatgtc gtgatacatc 8941 aacaacattt gatgaatggg ttgaacagtc agcaattgac taccaacata tgttgacata 9001 tttaaaaaca aacaacttcg aaaaagttgg ccaattaaca gaagcgaatg cattggctat 9061 gcatgcaact acaaagacag cgaatcctcc ttttagttac ctaactaaag aatcttatca 9121 agctatggaa gctgtaaaag aattgcgtca agaaggtttt gcatgctatt ttacaatgga 9181 tgcaggacct aatgtcaaag tactttgttt agagaaagat ttagcacaat tggcagagag 9241 attgggaaaa aattatcgaa ttatagtatc aaaaacaaaa gacttgccag acgtttaatt 9301 aggaggatga ttatttatga ctaatagaaa agatgatcat attaagtatg ctcttaaata 9361 ccaatcacct tataacgctt ttgatgatat tgaattaatt catcatagtc taccttctta 9421 tgatcttagt gacattgatt tatcaactca ctttgcagga caagattttg actttccatt 9481 ttatattaac gctatgacag gaggatctca aaaaggtaaa gctgttaatg aaaaattagc 9541 aaaagttgca gctgcaacag gtattgtaat ggtaacagga tcttatagtg cagctttgaa 9601 aaatccaaat gatgattctt atagattaca cgaggttgct gacaatctta aattagctac 9661 taacattggt ttagacaagc ctgttgcttt aggtcagcag acagtgcaag aaatgcaacc 9721 tttgtttctt caagtacatg ttaacgttat gcaagaattg ttaatgccag aaggtgaaag 9781 agtttttcat acttggaaaa aacatctagc tgaatatgct agtcaaatac cagtcccagt 9841 aattcttaaa gaagtaggct ttggtatgga tgttaactct attaaattag cacatgattt 9901 gggtattcaa actttcgata taagtggtag aggtggaact agtttcgctt atatagaaaa 9961 tcaacgtggt ggcgatagat catacttgaa tgactggggt caaactacag ttcagtgtct 10021 tttaaatgca caaggcctaa tggatcaagt cgaaatatta gcatcaggtg gtgtacgtca 10081 tcctcttgat atgattaaat gttttgtttt aggcgctcgt gcagtaggat tatctagaac 10141 ggtgttggaa ttagttgaaa aatatccaac tgagagagta atcgctatag taaatggttg 10201 gaaggaagaa ttgaaaataa ttatgtgtgc attggattgc aaaacaataa aagagctaaa 10261 aggcgtggat tatttattat acggacgact tcaacaagta aattaagcga agacttaatc 10321 attaggagga tgattattta tggcaggaac ggcatgggat actgatatac caacgcttat
21
SUBSTITUTE SHEET (RULE 26) 10381 ttcagtcaga gatgatgtgg atgcagtact tgaggacttt gttgatgttc aagagagagc 10441 ggcacacgga ccagagatgg ctccattgtt cacaacggct agaagacttt tgtttagtaa 10501 cggtaaaaga ctaagaccat tattatgcgt ggcggggtgg caagcagctg gagctccagg 10561 tgacccgcat gctgtcttgc gtgttgcagc agcattagaa ctattccaga cattcgcgct 10621 aatacatgac gacgtaatgg atgacagtga tactagaaga ggtagaccta gtgcacatag 10681 agctttggca gcagagtaca tgagtggcgg tggacgatta agtaaggctg aagcgcatgg 10741 cagaggagct gccattttgt taggtgacct tttattagtc tggtctgacg aaatgcttgc 10801 tggtgcgggc cttgaggcga gagcacgagc tagagtactt ccattagttg actctatgag 10861 agctgagtta gtatacggtc agtacttaga tttattatct actgatagaa tgtcaggtga 10921 cgttgaagcg gcgctaagag tagcaagata taagacaggc aagtatacag ttgaaagacc 10981 acttcatttg ggtgttagtt tagcgggcgg tgacccacga ttattacaga cgtgtacgga 11041 ttttgcattg ccattaggtg aagcattcca acttcgtgat gatcttttaa acgtctttgg 11101 ggatccagtt cgtactggta agccggttgg tgacgatata cgtgaaggta aagctacagt 11161 attgttagct cttgctatac aggacgcgtc agaagctgac ttaaaattat taggtacttt 11221 agttggtagt cctgagttag gtagtgaaga tatagaccga ttcagaacag taatggagac 11281 tacaggtgca cgtcaacgtg tagaggacat gataatcgca cgtagagaca ctgcacttgc 11341 aactttggaa gaagcggatt taccacagga tgtagcagag acattaagac agatcgtttg 11401 gatggcgacg gagagacaat cataacggat gtaaacagag aaacgtcaca ctggctcacc 11461 ttcgggtggg cctttctgcg tttatagcct aatacacctg ttcgtggaga atataaaaag 11521 ccagattatt aatccggctt ttttattatt t
In a further embodiment, the mosquito-repelling terpene comprises kolavelool. In one such embodiment, the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl-CoA C-acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS); TR51 15710 cyclase (CycAOl); and terpene synthase family (CycBOl). A list of the genes and encoded proteins are shown in Tables 6-7, and further details are provided in the examples.
Table 6. List of genes for the biosynthesis of Kolavelool.
Figure imgf000023_0001
22
SUBSTITUTE SHEET (RULE 26)
Figure imgf000024_0001
In another embodiment, the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of SEQ ID NO:2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO: 4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO:5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta-isomerase (IDI) comprising the amino acid sequence of SEQ ID NOT, geranylgeranyl pyrophosphate synthase (GGPS) comprising the amino acid sequence of SEQ ID NO: 14; TR51_15710 cyclase (CycAOl) comprising the amino acid sequence of SEQ ID NO:18; and terpene synthase family (CycBOl) comprising the amino acid sequence of SEQ ID NO: 19.
Table 7. Encoded polypeptides
Figure imgf000024_0002
23
SUBSTITUTE SHEET (RULE 26)
Figure imgf000025_0001
In one embodiment, the polynucleotides may be codon-optimized for expression in the bacterial cell of interest. In other embodiments, the bacterial cell may be transfected with separate polynucleotides encoding each polypeptide, with a single polynucleotide encoding all of the polypeptides, or 2 or more polynucleotides each encoding 1 one or more of the polypeptides. In one embodiment, the polynucleotides are arranged in a single operon, with all polypeptides encoded by a single polynucleotide.
In one such embodiment, the poly nucleotides encoding CycAOl and CycBOl are 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity. In another embodiment, the operon comprises, in 5’ to 3’ order, polynucleotides encoding CycAOl, CycBOl, atoB, HMGS, HMGR, MK, PMK, MVD, idi, and ggps.
In one embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotides encoding the polypeptides. In one such embodiment, the operon comprises a heterologous promoter 5’ to the polynucleotide CycAOl. Any heterologous promoter may be used as appropriate for an intended purpose. In various embodiments, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, or 25. In one embodiment, the heterologous promoter comprises the nucleotide sequence of SEQ ID NO:10.
In another embodiment, the operon comprises a heterologous insulator 5’ to the polynucleotide sequence encoding CycAOl. In a further embodiment, the heterologous
24
SUBSTITUTE SHEET (RULE 26) insulator is 3’ to the heterologous promoter. Any heterologous insulator may be used as appropriate for an intended purpose. In some embodiments, the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23. In one embodiment, the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11.
In a further embodiment, the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 10 of the genes encoding the polypeptides. Any heterologous RBS may be used as appropriate for an intended purpose. In one embodiment, the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
In another embodiment, the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO:20, as shown below.
SEQ ID NO:20
LOCUS Kolave lool_Pathw 11521 bp DNA
1 cggtggaaac gaggtcatca tttccttccg aaaaaacggt tgcatttaaa tcttacatat
61 gtaatacttt caaagactac atttgtaaga tttgaagctg tcaccggatg tgctttccgg
121 tctgatgagt ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa ttaggaggat
181 gattatttat gccagctatg agtgatgcgg acagaattgc agcattgtta aaagatcgag
241 ctgcagatcc agtcacgaag ttttcacctt ctccatacga aacgggtcaa tttttgcgaa
301 tttcagaacg tgcagatgtg ggcacgcctc aaattgatta tcttttagca acacaacgtc
361 cagacggatt atggggtagt gttggcttcg aattagttcc aactcttggt gctgttgctg
421 gtctttcttc aagacctgag tatgctgatc gtgctggtgt aacagacgca gttgctcgag
481 cttgtgaaaa gttatgggaa ttagcgttgg gcgaaggagg attaccaaaa ttgcctgata
541 cagttgcgtc agaaatcatt gtaccttctc ttattgactt gttaagtgaa gttttacaaa
601 gacatcgtcc agcagtggga ggtaaagcag gacaagaaca agagtttcca tcacctcctg
661 gagcaaaacc agagttatgg agacaattaa gtgatcgtat cgcacgtgga caagcaatcc
721 cgaaaactgc ttggcacaca ttagaagctt ttcatccatt acctaagcag tttgcagcta
781 ctgtaacacc agcagctgat ggtgctgtaa catgcagtcc ttcatctact gcagcgtggc
841 tatcagctgt tggtacagat gctggtgcgt ctacacgagc ttatttggat gaagcacaat
901 ctagatatgg tggtgcgatt cctatgggat caagtatgcc atattttgaa gtactatggg
961 tacttaatct agtcttaaaa tattttcctg atgttcctat acctagagaa ataattgaag
1021 aaattgctgc gggtttcagt gattctggta ttggtggtgg tccgggctta ccaccagatg
1081 gtgatgacac ggcatatgcg aacctagcag gagataaatt aggagcacct acacatccgg
1141 aaatattaat gaaattctgg gcggaagatc actttgttag ttatccaggt gaacaaacac
1201 catctgaaac tgtaaatgca catgcgctag aatatttaaa tcatcttcgt atgcgaagag
1261 gtataacaga attcggtgca gtggaagatg cgtgtgctga atgggtgata tcacaacaga
1321 ctgaggacgg ctgttggtac gataaatgga atgtctctcc ttactattca actgcagcat
1381 gtgtagaggc tttattagat gcacgtaaac aagatgaacc gcaattagac agtttacgtc
1441 gagctagaga gtggctttta agacatcaaa ctgatagtgg aggatggggt atggctgagc
SUBSTITUTE SHEET (RULE 26) 1501 cgagtcctga agaaacagct tatgctgtac tagcgcttga tttatttgca agtagaggtg
1561 gcgaaggagc tgaagaatgt gcagcagcta tttcacgtgc aaaagaattt tttactgatg
1621 aaagtagaga aaatcctcca ttatggatgg gcaaagattt atatacacca tttcgtattg
1681 ttgatgtaac tgttatgtgt ggaagagcag tcgttggtcg ttattaatta ggaggatgat
1741 tatttatgcc agacgctata gaatttgagc atgaaggtag aagaaaccca aactcagctg
1801 aagcagaatc agcttatagt tctattattg ctgcgttgga tttacaagaa tcagattatg
1861 ctgttatttc aggtcatagt cgtattgtgg gcgctgcagc tttagtttat cctgatgctg
1921 atgcggaaac tttacttgct gcaagtttat ggacggcttg tttaattgtg aatgatgaca
1981 gatgggatta tgtccaagag gatggaggcc gtttagcacc tggtgaatgg ttcgatggtg
2041 taacggaagt agttgatact tggcgaacag ctggtccaag actacctgat ccattctttg
2101 aattggttcg aacaacaatg tctagattgg acgcggctct tggagcagag gcagcagatg
2161 aaataggtca cgaaattaaa cgagcgataa cagcgatgaa atgggaagga gtttggaatg
2221 aatatacgaa gaaaactagt ttagcgactt atttaagttt tcgtcgtgga tattgtacaa
2281 tggatgttca agtggtatta gataaatgga ttaatggtgg tagatctttt gctgctttac
2341 gagatgaccc ggtcagaaga gcaatcgatg acgttgtagt acgttttgga tgcttatcta
2401 atgattacta ctcatggggc cgtgaaaaga aagcagtaga taaaagtaat gcagttagaa
2461 tccttatgga tcatgcaggt tatgatgaat caactgcgtt ggctcatgtt cgtgacgatt
2521 gtgtacaagc aattactgat cttgattgta ttgaagaaag tataaaacgt tctggacacc
2581 taggtagtca tgcacaggag ttactagatt atttagcatg tcatcgtcca ttaatctacg
2641 cagcagcaac atggcctaca gaaacaaatc gttatagata attaggagga tgattattta
2701 tggctgaagc ttatattgtt gaagcagtta gaacgcctgt cggtagaaga cgaggtggat
2761 tagctggcgt acacccagct gatttaggag cacatgcttt aacagctcta gttgcacgtt
2821 caggaattga tcctgcagcg gttgaagatg ttgtatttgg ttgcttagat acagttggtc
2881 cacaggctgg tgatattgca agaacttgtt ggttagcagc aggtcttcct gaagaagtac
2941 caggagtgac agtagataga caatgtggaa gttcacaaca agctgtgcat tttgctgcac
3001 aagctgttct atcaggtact caggatttgg tggtcgctgg aggagttcaa aatatgtcac
3061 aaattccaat agcatttgct tctagacaag cagctgaacc tttaggttta acgcaaggac
3121 cattcgcagg ttctgaaggt tggcgtgctc gatatggtga tcttccagtt aatcaatttc
3181 atggcgcaga actaattgcg gcaaagtggg gaattacacg acgtgatcaa gaagaatttg
3241 cattaagatc acatagaaga gcggtacgtg catctgacga aggtagattt gaccgagaaa
3301 cggttgcgta tggtaaagtc actgcggatg agggaccacg tagagataca agtttagaaa
3361 aaatggcggc tttagcacca gtaattgaag gtggaacgat tacagcagca tgttcaagtc
3421 aagtaagtga tggcgcagct gctatgttat tggcttcaga acgtgctttg agagaacatg
3481 gattaacgcc tagagcacgt gtgcatcatc taagtgtaag aggagaagat ccaattagaa
3541 tgttatctgc acctatccct gcaacagcac acgcattaaa aaaaactgct ttagcaattg
3601 atgatataga tttagtagaa atcaatgaag cgttcgcacc tgttgtactt gcatggttaa
3661 aagagactgg agcagatcct gaaaaagtga atgtcaatgg tggcgcaatc gctttaggac
3721 atcctttagg tgctacaggt gttagattga tgacgacatt attacacgag ttagaaagaa
3781 ctggtggtcg ttttggttta caaacaatgt gtgaaggagg aggccaggcg aatgttacga
3841 tcatagaaag actttagtta ggaggatgat tatttatgac tataggtata gacaaaatag
3901 gtttcgctac atctcaatat gttttaaaac tagaagattt agcattggca cgtcaagtag
26
SUBSTITUTE SHEET (RULE 26) 3961 atccagcaaa gttttctcaa ggtttattaa ttgaatcatt ttctgtagca cctatcacag
4021 aagatattat tacgttagca gcttctgcag cggaccaaat cctaactgat gaagatcgtg
4081 caaaaataga catggtaata ttagcaactg aaagtagtac tgaccaatca aaagcaagtg
4141 ctatttatgt tcaccatctt gtgggcattc aaccatttgc acgaagtttt gaagtaaagc
4201 aagcttgcta cagtgctaca gcagcattag attatgcaaa attacatgta gcaagtaaac
4261 cagatagtag agtattagtg atagcttcag acattgctcg ttatggagta ggatcaccag
4321 gagaaagtac acaaggaagt ggtagtattg cattgttggt gacagcaaat ccacgtatct
4381 tagcattaaa taaagataat gttgctcaaa cacgtgatat aatggatttc tggcgaccaa
4441 attactcatt tacaccatac gttgatggta tttatagtac taaacaatat cttaattgct
4501 tagagactac atggcaagct tatcagaaac gagaaaattt acaattaagt gattttgctg
4561 cagtctgttt tcatatccct tttcctaaat tagcattaaa gggtttaaat aacattatgg
4621 ataatactgt ccctccagag catagagaaa aactaattga agcgttccaa gcatcaatta
4681 cttattctaa acaaattggc aatatttata caggaagtct ttacttagga ttattgagtc
4741 ttttagaaaa tagtaaagtt ttacagtctg gtgacaagat cggttttttc tcatatggct
4801 caggagcagt ctctgaattt tattcaggcc aacttgttgc aggttatgat aaaatgttaa
4861 tgacaaatcg acaagcactt ttggatcaaa gaactagatt gtcagtttct aaatacgaag
4921 atttatttta tgaacaagtt caacttgatg ataatggtaa tgctaatttt gatatatatc
4981 taacaggtaa atttgctctt acagcaatca aagagcacca aagaatttac cacactaatg
5041 ataaaaatta attaggagga tgattattta tgactaaaac gaatttaaat tggtctggtt
5101 tctctaaaaa aacatttgaa gagcgtttac aattaataga aaaatttaaa ttactaaatg
5161 cagaaaactt aaatcaatta aaaacagatg ttttattacc tattcaaacg gcgaaccaga
5221 tgacagagaa tgtgttaggt cgtttagcat tacctttttc aatagcgcca gactttttag
5281 ttaatggttc tacataccaa atgccatttg tcacagaaga gccttctgtg gtagctgcag
5341 caagttttgc tgcaaagtta ataaaaagaa gtggtggatt taaagcgcaa acacttaatc
5401 gtcaaatgat cggccaaatc gtgttatacg atattgatca aatcgataat gcgaaagcag
5461 ctatcttaca taaaacgaaa aaattgatag ctttggctaa caaagcatat ccatcaattg
5521 ttaaacgagg tggtggcgca agaactattc atttggaaga gaaaggtgaa ttcttaatat
5581 tttatttaac ggtagacaca caagaagcaa tgggtgctaa tatggtcaat acaatgatgg
5641 aagctttggt tccagactta actcgtttga gtaagggaca ttgtttaatg gcaatattat
5701 caaattatgc tacagaatca ctagttacaa catcttgcga aataccagtt agattattag
5761 atcgtgacaa aacaaaatct ttacaattag cacaaaaaat tgaattagca agtcgtctag
5821 ctcaagtgga tccttatcgt gcaacaacac ataataaagg tatttttaat ggtatagacg
5881 ctgtcgtcat tgctactggt aacgactggc gtgcaattga agcaggtgca catgcatatg
5941 cttcacgaaa tggtagttat caaggcttaa gtcaatggca ttttgatcaa gacaaacagg
6001 ttttattagg tcaaatgaca ttacctatgc caattgcttc aaaaggaggt tcaataggct
6061 taaatcctac tgttagtatt gcacatgatt tattaaatca accagatgct aaaacactag
6121 ctcaacttat tgcaagtgta ggtttagcac aaaactttgc agcactaaaa gcgttaacta
6181 gttcaggtat ccaagcaggt catatgaaat tacacgcgaa atctcttgct ttattggcag
6241 gcgctactca agatgaaatt gcaccattgg taaacgcttt gcttgctgat aaaccaatta
6301 atctagaaaa agctcacttt tatttatcac aactacgtca aagttgagtc gacctgcagg
6361 catgcaagct tggctgtttt ggttaggagg atgattattt atgaacgaga atataggtta
27
SUBSTITUTE SHEET (RULE 26) 6421 tggaaaagca cattctaaaa ttattttaat aggtgaacat gcagtagtat atggctatcc
6481 agcaattgct cttcctctta cagatataga agtagtgtgt catatttttc cagcggataa
6541 accactagtt tttgacttct atgacacttt aagtacagca atatatgcgg cacttgatta
6601 tcttcaaaga ttgcaagaac caatagctta cgaaatagtt agtcaagtac cacagaagcg
6661 tggcatgggt agtagtgctg ctgttagtat tgcagcgatt agagctgttt tttcttattg
6721 tcaagagcct ttaagtgacg atttacttga aatcctagta aataaagcag aaataattgc
6781 acacacaaat ccttcaggat tagatgctaa aacatgctta tctgatcacg ctataaaatt
6841 tattagaaat ataggttttg agacaatcga aattgcttta aatggttatc taattattgc
6901 tgatacgggt attcatggtc atacaagaga agctgttaat aaagtcgctc agtttgaaga
6961 aacaaattta ccatatttag caaaacttgg tgcattaaca caagcattag aaagagctat
7021 caaccaaaaa aataaggtag caattggaca attaatgaca caagctcata gtgcattgaa
7081 agcaattggt gtatctatct ctaaagctga tcaattagtc gaagcagcat tacgagcagg
7141 tgcattaggt gctaaaatga caggtggtgg tctaggtggt tgtatgattg ctttagcaga
7201 tacaaaagat atggcagaaa aaatttcaca tagattaaag gaggaaggag cggtaaatac
7261 atggattcag atgttatagt taggaggatg attatttatg agtaactact gtgtacaaac
7321 gggcggtaaa ttatatttaa caggagaata tgcaatactt attccaggtc aaaaagcttt
7381 aattcacttt attccattaa tgatgactgc tgaaatcagt ccagctgcac atattcaact
7441 tgcttcagat atgttttcac acaaagcggg tatgacacct gatgcaagtt atgctttaat
7501 tcaagcaaca gttaaaacat ttgcagatta cctaggacaa agtattgatc aattagaacc
7561 tttcagtctt attatcacgg gtaaaatgga acgtgatgga aaaaaattcg gtataggaag
7621 ttcaggatct gttactttat taactttaaa agcattgtca gcttattatc aaatcacatt
7681 aacaccagaa ttgttattta aattagctgc atatactttg ctaaaacagg gagataacgg
7741 ttctatggga gacatagcgt gcattgctta ccaaacatta gttgcatata catcttttga
7801 tcgtgaacaa gtgtcaaatt ggttacaaac tatgccatta aaaaaacttt tagtcaaaga
7861 ttggggatat catattcaag taattcagcc tgctttgcct tgtgatttct tagttggttg
7921 gactaagata cctgcaattt cacgtcaaat gattcagcaa gtaacggctt ctattacacc
7981 tgcgttttta cgtacgtcat atcaattaac acaatctgct atggtggctt tacaagaagg
8041 ccataaagaa gaattaaaaa aatcattggc tggagcttca cacttattaa aagaattaca
8101 ccctgctatt tatcatccta aattggttac attagttgct gcttgtcaaa aacaggatgc
8161 agtagcaaaa agtagtggaa gtggtggagg cgactgcggt attgctcttg catttaatca
8221 agatgcgcgt gatacattaa tttcaaaatg gcaagaagct gatatagctc ttttatacca
8281 agaaagatgg ggagagaatg actagttagg aggatgatta tttatggatc ctaatgtaat
8341 cacggttact tcatatgcaa atatagcaat aattaaatac tggggtaaag aaaatcaagc
8401 taaaatgatc ccttctacat cttctatttc attgacatta gaaaatatgt tcactactac
8461 ttcagtgtca tttttaccag acactgcaac ttcagatcaa ttttatataa acggtgtgtt
8521 acaaaatgat gaagaacata cgaaaatatc aacaattatt gatcaattcc gtcaaccagg
8581 acaagctttt gtaaaaatgg aaactcaaaa taacatgcct acagctgcag gattatcatc
8641 atcatcttca ggtttgtctg cattagtaaa ggcttgtgat caattgtttg atacacagct
8701 agatcaaaaa gcattagcac aaaaagcaaa atttgcgtct ggaagttcat cacgttcttt
8761 ctttggtcct gtcgcggcat gggataaaga ttctggcgca atatataaag ttgaaacgga
8821 cttgaaaatg gcgatgatta tgttagtttt aaacgcagca aaaaaaccta tttcatcacg
28
SUBSTITUTE SHEET (RULE 26) 8881 tgaaggtatg aaattatgtc gtgatacatc aacaacattt gatgaatggg ttgaacagtc 8941 agcaattgac taccaacata tgttgacata tttaaaaaca aacaacttcg aaaaagttgg 9001 ccaattaaca gaagcgaatg cattggctat gcatgcaact acaaagacag cgaatcctcc 9061 ttttagttac ctaactaaag aatcttatca agctatggaa gctgtaaaag aattgcgtca 9121 agaaggtttt gcatgctatt ttacaatgga tgcaggacct aatgtcaaag tactttgttt 9181 agagaaagat ttagcacaat tggcagagag attgggaaaa aattatcgaa ttatagtatc 9241 aaaaacaaaa gacttgccag acgtttaatt aggaggatga ttatttatga ctaatagaaa 9301 agatgatcat attaagtatg ctcttaaata ccaatcacct tataacgctt ttgatgatat 9361 tgaattaatt catcatagtc taccttctta tgatcttagt gacattgatt tatcaactca 9421 ctttgcagga caagattttg actttccatt ttatattaac gctatgacag gaggatctca 9481 aaaaggtaaa gctgttaatg aaaaattagc aaaagttgca gctgcaacag gtattgtaat 9541 ggtaacagga tcttatagtg cagctttgaa aaatccaaat gatgattctt atagattaca 9601 cgaggttgct gacaatctta aattagctac taacattggt ttagacaagc ctgttgcttt 9661 aggtcagcag acagtgcaag aaatgcaacc tttgtttctt caagtacatg ttaacgttat 9721 gcaagaattg ttaatgccag aaggtgaaag agtttttcat acttggaaaa aacatctagc 9781 tgaatatgct agtcaaatac cagtcccagt aattcttaaa gaagtaggct ttggtatgga 9841 tgttaactct attaaattag cacatgattt gggtattcaa actttcgata taagtggtag 9901 aggtggaact agtttcgctt atatagaaaa tcaacgtggt ggcgatagat catacttgaa 9961 tgactggggt caaactacag ttcagtgtct tttaaatgca caaggcctaa tggatcaagt 10021 cgaaatatta gcatcaggtg gtgtacgtca tcctcttgat atgattaaat gttttgtttt 10081 aggcgctcgt gcagtaggat tatctagaac ggtgttggaa ttagttgaaa aatatccaac 10141 tgagagagta atcgctatag taaatggttg gaaggaagaa ttgaaaataa ttatgtgtgc 10201 attggattgc aaaacaataa aagagctaaa aggcgtggat tatttattat acggacgact 10261 tcaacaagta aattaagcga agacttaatc attaggagga tgattattta tggcaggaac 10321 ggcatgggat actgatatac caacgcttat ttcagtcaga gatgatgtgg atgcagtact 10381 tgaggacttt gttgatgttc aagagagagc ggcacacgga ccagagatgg ctccattgtt 10441 cacaacggct agaagacttt tgtttagtaa cggtaaaaga ctaagaccat tattatgcgt 10501 ggcggggtgg caagcagctg gagctccagg tgacccgcat gctgtcttgc gtgttgcagc 10561 agcattagaa ctattccaga cattcgcgct aatacatgac gacgtaatgg atgacagtga 10621 tactagaaga ggtagaccta gtgcacatag agctttggca gcagagtaca tgagtggcgg 10681 tggacgatta agtaaggctg aagcgcatgg cagaggagct gccattttgt taggtgacct 10741 tttattagtc tggtctgacg aaatgcttgc tggtgcgggc cttgaggcga gagcacgagc 10801 tagagtactt ccattagttg actctatgag agctgagtta gtatacggtc agtacttaga 10861 tttattatct actgatagaa tgtcaggtga cgttgaagcg gcgctaagag tagcaagata 10921 taagacaggc aagtatacag ttgaaagacc acttcatttg ggtgttagtt tagcgggcgg 10981 tgacccacga ttattacaga cgtgtacgga ttttgcattg ccattaggtg aagcattcca 11041 acttcgtgat gatcttttaa acgtctttgg ggatccagtt cgtactggta agccggttgg 11101 tgacgatata cgtgaaggta aagctacagt attgttagct cttgctatac aggacgcgtc 11161 agaagctgac ttaaaattat taggtacttt agttggtagt cctgagttag gtagtgaaga 11221 tatagaccga ttcagaacag taatggagac tacaggtgca cgtcaacgtg tagaggacat 11281 gataatcgca cgtagagaca ctgcacttgc aactttggaa gaagcggatt taccacagga
29
SUBSTITUTE SHEET (RULE 26) 11341 tgtagcagag acattaagac agatcgtttg gatggcgacg gagagacaat cataacggat
11401 gtaaacagag aaacgtcaca ctggctcacc ttcgggtggg cctttctgcg tttatagcct
11461 aatacacctg ttcgtggaga atataaaaag ccagattatt aatccggctt ttttattatt
11521 t
In another aspect, the disclosure provides polynucleotides comprising the nucleotide sequence of any one of SEQ ID NO: 13, 17, and 20. In another embodiment, the disclosure provides recombinant expression vectors comprising the polynucleotide "Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. "Control sequences" operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence is still considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, etc. Such expression vectors can be of any type known in the art In another embodiment, the present disclosure provides cells comprising the polynucleotide of the disclosure, such as a prokaryotic cell.
In a further embodiment, the disclosure provides compositions comprising a plurality of the engineered bacterial cell of any previous claim and a pharmaceutically acceptable carrier. The compositions may be of any suitable format for application of the compositions to the skin of a subject, including but not limited to a human subject. In one embodiment, the composition is formulated for topical application. In various non-limiting embodiments, the topical formulation may be in the form of a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
The disclosure also provides methods to repel mosquitos, comprising contacting the skin of a subject with a plurality of the engineered bacterial cells of any embodiment or combination of embodiments herein, or with the composition of any embodiment or combination of embodiments herein, thereby repelling mosquitos from skin of the subject.
In another aspect, the disclosure provides methods to repel mosquitos, comprising contacting the skin of a subject with kaur- 16-ene, kolavelool, or a combination thereof, thereby repelling mosquitos from skin of the subject.
30
SUBSTITUTE SHEET (RULE 26) In these methods, the subject may be any subject that can benefit from repelling mosquitos, including but not limited to human subjects. The compositions or compounds may be administered in any suitable formulation, including being formulated for topical application. In vanous non-limiting embodiments, the topical formulation may be in the form of a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
Examples
Engineering human skin microbes to produce mosquito repellent terpenes.
Mosquitoes are a major source of vector-bome diseases including malaria, dengue, West Nile, zika and yellow fever, affecting millions every year. Vector-bome diseases account for 17% of all infectious diseases, resulting in >700,000 deaths annually (World Health Organization, 2022). Minimizing contact of mosquitoes is key to preventing the transmission of illnesses. Currently, there are several strategies for repelling mosquitoes: (i) physical barriers such as nets, (ii) Eliminating sites that are conducive for mosquito breeding, longevity and parasite sporogony such as ponds, swamps marshes and other bodies of stillwater (iii) Pesticides and chemical sprays containing anti-mosquito compounds, of which the current gold standard, N, N-diethyl-meta-toluarmde (DEET), can be found in over-the- counter sprays such as OFF! ®. While DEET is a highly effective mosquito repellent, it requires consistent re-application due to its relatively short effective period and is cost prohibitive to much of the developing world.
To mitigate disease transmission by mosquitoes, here we propose engineering bacteria on the surface of human skin to produce mosquito repellent terpenes. Terpenes are a structurally diverse class of natural products comprising of over 70,000 compounds. Terpenes are produced by plants, fungi and bacteria, act as pheromones and are involved in a number of important ecological functions such as plant cell signaling and defense. To screen for terpenes that are mosquito repellents, we first used a mosquito olfaction apparatus that can quantify the repellency of terpenes that are generated by Escherichia coll strains heterologously expressing a number of different terpene pathways (Figure 1). The olfactometer used here is a dual-choice apparatus for screening the behavior of molecules that contribute to mosquito repulsion (Figure 2A). This behavioral assay identified two terpenes as mosquito repellents: the monoterpene eucalyptol (also known as 1,8 cineole) and a diterpene repellent kaur-16-ene (Figure 2B).
With this promising result, we moved forward with the complex task of integrating the biosynthetic pathways of eucalyptol and kaur-16-ene into the genome of a primary skin 31
SUBSTITUTE SHEET (RULE 26) isolate, Staphylococcus epidermidis LM088. Manipulation of skin commensals most commonly found on humans has been particularly challenging, with only a handful of reported examples to date. These reported examples often involve introducing a single gene by plasmid transformation, which is not suitable for expressing & maintaining the multi-gene biosynthetic pathways required for terpene production. To address this challenge, we used a conjugation strategy optimized in the Voigt lab to integrate exogenous DNA in undomesticated bacteria (Voigt, Nat. Microbiol. 2018, 3, 9). This strategy, referred to as mini-ICE (integrative-conjugative elements), is able to integrate >100 Kb of DNA in undomesticated soil bacteria. Using this Bacillus subtilis-based conjugation strategy , the pathways of eucalyptol (sMIH197, S. epidermidis + eucalyptol, Figure 3A) and kaur-16-ene (sMIH210, S. epidermidis + kaur-16-ene, Figure 4A) were successfully integrated into the genome of an undomesticated primary skin isolate S. epidermidis LM088. Analysis of the engineered strains by solid-phase microextraction gas chromatography mass spectrometry' (SPME-GC-MS) confirmed that sMIH197 and sMIH210 successfully produced eucalyptol (Figure 3B) and kaur-16-ene (Figure 4B), respectively This is the first example of a primary skin isolate heterologously expressing terpene biosynthetic pathways and successfully generating terpenes of interest.
The mosquito olfactometer was then used to assess the efficacy of these engineered, terpene producing S. epidermidis strains as mosquito repellents. In vitro analysis of these engineered strains in an olfactometer showed that the eucalyptol-producing sMIH197 readily repels mosquitoes (Figure 3C). This is the first demonstration of an engineered skin microbe successfully repelling mosquitoes. Ultimately, manipulating the skin microbiome to produce mosquito repellent terpenes such as eucalyptol and kaur-16-ene offers a novel strategy of repelling mosquitoes and minimizing transmission of mosquito associated illnesses.
Mini-ICEBsl conjugation of S. epidermidis LM088 to generate terpene producing skin commensals
This B. subtilis-based conjugation strategy that was used in this disclosure was developed in the Voigt lab. More details can be found here: Voigt et al, Nat. Microbiol. 2018, 3, 9. The conjugation protocol was described in the 2018 manuscript with the following modifications for the integration of multi-gene terpene pathways in 5. epidermidis'.
Donor B. subtilis strains were generated by natural transformation of integrative plasmids. A single colony of B. subtilis was inoculated in 2ml of LB + 100 pg ml 1 D-alanine
32
SUBSTITUTE SHEET (RULE 26) and grown at 37 °C and 250 r.p.m. until optical density at 600 nm (OD600) =1. Cultures were then back-diluted 20-fold into minimal Davis (MD) medium (lx phosphatecitrate (PC) buffer (per liter: 10.7 g K2HPO4, 6 g KH2PO4, 1 g NasCeHsO?, adjusted to pH 7.5 with KOH), 2% glucose, 3mM MgSOr, 0.02mM CeHsFeNO?, 14.5mM C4H6KNO4). 0.2 mL of the MD medium was mixed 0.02 mL of B. subtilis at OD600=1, 1000 ng of plasmid DNA (containing the full terpene pathway subcloned in to pJAB980), 50 pg ml 1 tryptophan, 50 pg ml 1 phenylalanine, and 100 pg ml 1 D-alanine. Cells were grown for 24 h at 37 °C and 250 r.p.m and were plated onto LB selective medium containing 100 pg ml 1 D-alanine and 5 pg ml 1 of tetracycline. Successful B. subtilis colony donor strains were screened by PCR and used for conjugating primary skin commensals.
Donor (B. subtlis + terpene pathway) and recipient (S', epidermidis LM088) strains were picked from solid medium and inoculated in 2 mL of medium in 15 ml culture tube for 12 h at 37°C, 250 r.p.m. The dense overnight cultures were diluted 100-fold into 25 mL of medium in 250 mL Erlenmeyer flasks and grown at 37°C, 250 r.p.m. The recipient S. epidermidis LM088 strain was grown to an OD600 of 1. The donor B. subtilis strain was grown to OD600=0.35, after which 1% xylose (w/v) was added to the culture to induce ICE conjugation machinery and was further grown to an OD600=1, at which 1.5 mL of Donor and 1.5 mL of recipient were mixed and filtered onto sterile nitrocellulose membranes. Filters were placed on Petri dishes containing Tris-Spizzizen Salts (TSS) (per liter: 2 g NH4Q, 0.35 g K2HPO4*3H2O, 6 g Tns base; pH to 7.5 with HC1) with 125 mM MgCh and 2% agar. Matings were incubated for 5 h at 37 °C. Cells were then rinsed off the filter using 3ml TSS and plated on BHI + agar selective medium with 2.5 pg ml 1 of tetracycline. Successful S. epidermidis LM088 transconjugants were screened by colony PCR using primers specific to the terpene pathway of interest.
Details of genes used for the production of terpenes in S. epidermidis LM088
All genes related to terpene production were codon optimized using the codon usage shown in Table 8.
Table 8. Codon usage table
33
SUBSTITUTE SHEET (RULE 26) Stapftytoeoccas epiderinidis ATCC 12228 [gbbd]: 2485 CDS's (714252 codons)
Figure imgf000035_0001
34
SUBSTITUTE SHEET (RULE 26)

Claims

We claim
1. An engineered bacterial cell for producing a mosquito-repelling terpene, comprising an engineered terpene biosynthetic pathway introduced into a bacterial cell that is not otherwise capable of producing the terpene, wherein the engineered bacterial cell is capable of producing the mosquito-repelling terpene.
2. The engineered bacterial cell of claim 1, wherein the bacterial cell is a human skin microbe.
3. The engineered bacterial cell of claim 1, wherein the bacterial ceil is Corynebacterium amycolatum, Staphylococcus caprae, or Staphylococcus epidermidis .
4. lire engineered bacterial cell any one of claims 1-3, wherein the bacterial cell is Staphylococcus epidermidis .
5. Tiie engineered bacterial cell of any one of claims 1-4, wherein the bacterial cell is
Corynebacterium amycolatum .
6. The engineered bacterial cell of any one of claims 1-5, wherein the mosquitorepelling terpene is selected from the group consisting of eucalyptol, kaur- 16-ene, and kolavelool.
7. The engineered bacterial cell of any one of claims 1-5, wherein the mosquitorepelling terpene comprises eucalyptol.
8. The engineered bacterial cell of claim 7, wherein the bacterial cell is transformed with polynucleotides encoding tire following polypeptides having enzymatic activity: acetyl-CoA C -acetyltransferase (AtoB), hydroxymethyiglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranyldiphosphate synthase (GPPS), and 1,8 cineole synthase (bCinS).
9. The engineered bacterial cell of claim 8, wherein the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1 , hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of SEQ ID NO:2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the ammo acid sequence of SEQ ID NO:3, mevalonate kinase (MK.) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO:5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta- isomerase (IDI) comprising the ammo acid sequence of SEQ ID NO: 7, geranyldiphosphate synthase (GPPS) comprising the amino acid sequence of SEQ ID NO: 8, and 1,8 cineole synthase (bCinS) comprising the amino acid sequence of SEQ ID NO: 9.
10. The engineered bacterial cell of any one of claims 8-9, wherein the polynucleotides are codon-optimized for expression in the bacterial cell.
11. The engineered bacterial ceil of any one of claims 8-10, wherein the polynucleotides are arranged in a single operon.
12. The engineered bacterial cell of claim 11, wherein the polynucleotide encoding bCinS is 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity.
13. The engineered bacterial cell of claim 11 or!2, wherein die operon comprises, m 5’ to 3’ order, polynucleotides encoding bCinS, atoB, HMGS, HMGRJMK, PMK, MVD, idi, and gPPS-
14. The engineered bacterial cell of any one of claims 1 1-13, wherein the operon comprises a heterologous promoter 5" to the polynucleotide sequence encoding bCinS.
15. The engineered bacterial cell of claim 14, wherein the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, o 25.
16. The engineered bacterial ceil of any one of claims 1 1 -15, wherein the operon comprises a heterologous insulator 5’ to the polynucleotide encoding bCinS.
17. The engineered bacterial cell of claim 16, wherein the heterologous insulator is 3’ to the heterologous promoter.
18. The engineered bacterial cell of claim 16 or 17, wherein the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23.
19. The engineered bacterial cell of any one of claims 9-18, wherein the polynucleotides comprise a heterologous ribosome binding site (RBS) 5’ to all 9 of the genes encoding the polypeptides.
20. The engineered bacterial cell of claim 19, wherein the RBS comprises the nucleotide sequence of SEQ ID NO: 12
21. The engineered bacterial cell of any one of claims 1-20, wherein the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13.
22. lire engineered bacterial cell of any one of claims 1-5, wherein tire mosquitorepelling terpene comprises kaur-l 6-ene.
23. The engineered bacterial cell of claim 22, wherein the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: wherein the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl -Co A C-acetyltransferase (AtoB), hydroxymethylglutaryl- CoA synthase (HMGS), hydroxymethylglutaiyl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (IDI), geranylgeranyl pyrophosphate synthase (GGPS), PtmT2 cyclase (CycA07), and Hypothetical cyclase (CycB04)
24. The engineered bacterial cell of claim 23, wherein the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1, hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of SEQ ID NO:2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising tire amino acid sequence of SEQ ID NO:5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta- isomerase (IDI) comprising the amino acid sequence of SEQ ID NO: 7; geranylgeranyl pyrophosphate synthase (GGPS) comprising the ammo acid sequence of SEQ ID NO: 14; PtmT2 cyclase (CycA07) comprising the amino acid sequence of SEQ ID NO: 15; and Hypothetical cyclase (CycB04) comprising the amino acid sequence of SEQ ID NO: 16.
25. The engineered bacterial cell of any one of claims 23-24, w herein the polynucleotides are codon-optimized for expression in the bacterial cell.
26. The engineered bacterial cell of any one of claims 23-25, wherein the polynucleotides are arranged into a single operon.
27. The engineered bacterial cell of claim 26, wherein the polynucleotides encoding
CycA07 and CycB04 are 5’ in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity.
2.8. The engineered bacterial cell of claim 26 or 27, wherein the operon comprises, in 5’ to 3’ order, polynucleotides encoding CycA07, CycB04, atoB, HMGS, HMGR,MK, PMK, MVD, PMD, idi, and ggps.
29. The engineered bacterial cell of any one of claims 26-28, wherein the operon comprises a heterologous promoter 5’ to polynucleotide encoding CycA07.
30. The engineered bacterial cell of claim 29, wherein the heterologous promoter comprises the nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, o 25.
31. The engineered bacterial cell of any one of claims 26-30, wherein the first operon comprises a heterologous insulator 5’ to the polynucleotide encoding CycA07.
32. The engineered bacterial cell of claim 31 , wherein a heterologous insulator is 3’ to the heterologous promoter and 5’ to the polynucleotide encoding CycA07.
33. The engineered bacterial cell of claim 31 or 32, wherein the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 11 or 23.
34. The engineered bacterial cell of any one of claims 23-33, wherein the polynucleotides comprise a heterologous RBS 5’ to all 10 of the polynucleotide encoding tire polypeptides.
35. The engineered bacterial cell of claim 34, wherein the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
36. Hie engineered bacterial cell of any one of claims 22-35, wherein the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17
37. The engineered bacterial cell of any one of claims 1-5, wherein the mosquitorepelling terpene comprises kolavelool.
38. The engineered bacterial cell of claim 37, wherein the bacterial cell is transformed with polynucleotides encoding the following polypeptides having enzymatic activity: acetyl- CoA C-acetyltransferase (AtoB), hydroxymetbylghitaryl-CoA synthase (HMGS), hydroxymethylghitaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), Isopentenyl-diphosphate Delta-isomerase (1DI), geranylgeranyl pyrophosphate synthase (GGPS); TR51_15710 cyclase (CycAOl); and terpene synthase family (CycBOl).
39. The engineered bacterial cell of claim 38, wherein the bacterial cell is transformed with polynucleotides encoding acetyl-CoA C-acetyltransferase (AtoB) comprising the amino acid sequence of SEQ ID NO: 1 , hydroxymethylglutaryl-CoA synthase (HMGS) comprising the amino acid sequence of SEQ ID NO: 2, hydroxymethylglutaryl-CoA reductase (HMGR) comprising the amino acid sequence of SEQ ID NO:3, mevalonate kinase (MK) comprising the amino acid sequence of SEQ ID NO:4, phosphomevalonate kinase (PMK) comprising the amino acid sequence of SEQ ID NO:5, diphosphomevalonate decarboxylase (MVD) comprising the amino acid sequence of SEQ ID NO:6, Isopentenyl-diphosphate Delta- isomerase (IDI) comprising the amino acid sequence of SEQ ID NO:7, geranylgeranyl pyrophosphate synthase (GGPS) comprising the amino acid sequence of SEQ ID NO: 14; TR51_15710 cyclase (CycAOl) comprising the amino acid sequence of SEQ ID NO:18; and terpene synthase family (CycBOl) comprising the amino acid sequence of SEQ ID NO: 19,
40. lire engineered bacterial cell of any one of claims 38-39, wherein the polynucleotides are codon-optimized for expression in the bacterial cell,
41 . The engineered bacteria! cell of any one of claims 38-40, wherein the polynucleotides are arranged into a single operon.
42. The engineered bacterial cell of claim 41, wherein the polynucleotides encoding CycAO 1 and CycBO 1 are 5 ' in the operon relative to polynucleotides encoding the other polypeptides having enzymatic activity.
43. Hie engineered bacterial cell of claim 41 or 42, wherein the operon comprises, in 5’ to 3’ order, polynucleotides encoding CycAOl, CycBOl, atoB, HAIGS, HMGR, MK, PMK, MVD , idi, and ggps.
44. lire engineered bacterial cell of any one of claims 41-43, wherein the operon comprises a heterologous promoter 5’ to the polynucleotide sequence encoding CycAOl .
45. The engineered bacterial cell of claim 44, wherein the heterologous promoter comprises tire nucleotide sequence of SEQ ID NO: 10, 21, 22, 24, o 25.
46. The engineered bacterial cell of any one of claims 41-45, wherein the first operon comprises a heterologous insulator 5’ to the polynucleotide encoding CycAOl.
47. The engineered bacterial cell of claim 46, wherein the heterologous insulator is 3’ to the heterologous promoter.
48. The engineered bacterial cell of claim 46 or 47, wherein the heterologous insulator comprises the nucleotide sequence of SEQ ID NO: 1 1 or 23.
49. The engineered bacterial ceil of any one of claims 38-48, wherein the polynucleotides comprise a heterologous RBS 5’ to all 10 of the polynucleotides encoding the polypeptides.
50 The engineered bacterial cell of claim49, wherein the RBS comprises the nucleotide sequence of SEQ ID NO: 12.
51 . The engineered bacterial cell of any one of claims 37-50, wherein the bacterial cell is transformed with the polynucleotide comprising the nucleotide sequence SEQ ID NO:20.
52. A polynucleotide comprising the nucleotide sequence of any one of SEQ ID NO: 13, 17, and 20.
53. A recombinant expression vector comprising the polynucleotide of claim 52.
54. A recombinant host cell comprising the polynucleotide or recombinant expression vector of claims 52-53.
55. A composition comprising a plurality of the engineered bacterial cell of any previous claim and a pharmaceutically acceptable carrier.
56. The composition of claim 55, wherein the composition is formulated for topical administration, including but not limited to a spray, a lotion, an oil, an ointment, a cream, a balm, a foam, a gel, or a salve.
57. A method to repel mosquitos, comprising contacting the skin of a subject with a plurality of the engineered bacterial cells of any preceding claim, or with the composition of any preceding claim, thereby repelling mosquitos from skin of the subject.
58. A method to repel mosquitos, comprising contacting the skin of a subject with kaur- 16-ene, kolavelool, or a combination thereof, thereby repelling mosquitos from skin of the subject.
59. The method of claim 58, wherein the kaur-16-ene, kolavelool, or combination thereof, is applied to the skin as a topical formulation.
PCT/US2024/023221 2023-06-08 2024-04-05 Engineering human skin microbes to produce mosquito repellent terpenes Ceased WO2024253742A1 (en)

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Citations (5)

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US20180037912A1 (en) * 2014-01-31 2018-02-08 University Of Copenhagen Methods for Producing Diterpenes
US20190352647A1 (en) * 2017-02-27 2019-11-21 Sekisui Chemical Co., Ltd. Recombinant cell, method for producing recombinant cell, and method for producing isoprene or terpene
CN110538082A (en) * 2019-09-17 2019-12-06 江西农业大学 Efficient mosquito repelling method by associating terpenoid repellent and attractant supermolecule
US20200056145A1 (en) * 2017-04-17 2020-02-20 The Regents Of The University Of California Engineered commensal bacteria and methods of use
WO2023133490A1 (en) * 2022-01-07 2023-07-13 Ginkgo Bioworks, Inc. Skin commensal bacteria engineered to produce terpenes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180037912A1 (en) * 2014-01-31 2018-02-08 University Of Copenhagen Methods for Producing Diterpenes
US20190352647A1 (en) * 2017-02-27 2019-11-21 Sekisui Chemical Co., Ltd. Recombinant cell, method for producing recombinant cell, and method for producing isoprene or terpene
US20200056145A1 (en) * 2017-04-17 2020-02-20 The Regents Of The University Of California Engineered commensal bacteria and methods of use
CN110538082A (en) * 2019-09-17 2019-12-06 江西农业大学 Efficient mosquito repelling method by associating terpenoid repellent and attractant supermolecule
WO2023133490A1 (en) * 2022-01-07 2023-07-13 Ginkgo Bioworks, Inc. Skin commensal bacteria engineered to produce terpenes

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