阅读说明：本技术 基因改造植物对室内空气污染物的降解 (Degradation of indoor air pollutants by genetically modified plants ) 是由 张龙 S·E·斯特兰德 于 2019-12-18 设计创作，主要内容包括：公开了能够降低发达国家城市家庭室内空气中挥发性有机致癌化合物诸如甲醛、苯和氯仿水平的基因改造室内植物。该植物表达解毒转基因哺乳动物细胞色素P450 2e,并且显示出充分的对苯和氯仿的解毒活性。还公开了利用该植物的空气净化生物过滤器及其用途。(Genetically modified houseplants capable of reducing the levels of volatile organic carcinogenic compounds such as formaldehyde, benzene and chloroform in the indoor air of urban households in developed countries are disclosed. The plant expresses detoxified transgenic mammalian cytochrome P4502 e and exhibits sufficient detoxifying activity against benzene and chloroform. Also discloses an air purification biological filter using the plant and the use thereof.)

1. A transgenic houseplant, houseplant part, or houseplant cell stably transformed with an expression cassette comprising a first polynucleotide sequence encoding a mammalian cytochrome protein, wherein the transgenic houseplant is capable of removing volatile organic compounds from the air.

2. The transgenic houseplant, houseplant part or houseplant cell according to claim 1, wherein the expression cassette further comprises a second polynucleotide sequence encoding a visual selection marker.

3. The transgenic houseplant, houseplant part or houseplant cell according to claim 1 or claim 2, wherein the first polynucleotide sequence is operably linked to a first promoter functional in the transgenic houseplant.

4. The transgenic houseplant, houseplant part or houseplant cell according to claim 2 or claim 3, wherein the second polynucleotide sequence is operably linked to a second promoter functional in the transgenic houseplant.

5. The transgenic plant, plant part, or plant cell of any one of claims 1-4, wherein the transgenic house plant is a transgenic scindapsus aureus.

6. The transgenic houseplant, houseplant part, or houseplant cell according to any one of claims 1-5, wherein said mammalian cytochrome protein is cytochrome P450 or a variant or homolog thereof having at least about 90% amino acid sequence homology thereto.

7. The transgenic house plant, house plant part or house plant cell according to any one of claims 1-6, wherein said mammalian cytochrome protein is cytochrome P4502E 1 or a variant thereof or a homologue thereof having at least about 90% amino acid sequence homology thereto.

8. The transgenic houseplant, houseplant part or houseplant cell according to claim 7, wherein said cytochrome P4502E 1 is rabbit cytochrome P4502E 1 or a variant or homolog thereof having at least about 90% amino acid sequence homology thereto.

9. A transgenic houseplant, houseplant part or houseplant cell according to any one of claims 1 to 8, wherein the polynucleotide sequence encoding the mammalian cytochrome protein is codon optimized for the plant.

10. The transgenic houseplant, houseplant part or houseplant cell according to claim 9, wherein the polynucleotide sequence encoding the mammalian cytochrome protein is codon optimized for monocots.

11. The transgenic houseplant, houseplant part or houseplant cell according to any one of claims 2 to 10, wherein the visual selectable marker is a fluorescent protein.

12. The transgenic houseplant, houseplant part, or houseplant cell according to claim 11, wherein the fluorescent protein is green fluorescent protein mGFP-ER, eGFP, or a variant or homolog thereof having at least about 90% amino acid sequence homology thereto.

13. The transgenic houseplant, houseplant part, or houseplant cell according to any one of claims 1-12, wherein the expression cassette further comprises a third polynucleotide sequence encoding a positive selection marker.

14. The transgenic houseplant, houseplant part or houseplant cell according to claim 13, wherein the positive selection marker is a protein that confers antibiotic resistance to the transgenic houseplant.

15. The transgenic houseplant, houseplant part or houseplant cell according to claim 14, wherein said positive selection marker is hygromycin B phosphotransferase or a variant thereof or a homolog thereof having at least about 90% amino acid sequence homology thereto.

16. The transgenic houseplant, houseplant part or houseplant cell according to any one of claims 1 to 15, wherein the expression cassette further comprises a fourth polynucleotide sequence encoding an aldehyde dehydrogenase.

17. The transgenic house plant, house plant part or house plant cell of claim 16 wherein the aldehyde dehydrogenase is formaldehyde dehydrogenase (FALDH) or a variant thereof or a homolog thereof having at least about 90% amino acid sequence homology thereto.

18. A transgenic houseplant, houseplant part or houseplant cell according to any one of the preceding claims, wherein the volatile organic compound is selected from the group consisting of: 1, 4-dichlorobenzene, benzene, 1, 3-butadiene, formaldehyde, acetaldehyde, naphthalene, acrylonitrile, carbon tetrachloride, dichlorobromomethane, dibromochloromethane, bromoform, trichloroethylene, vinyl chloride, methylchloroform, cis-1, 2-dichloroethylene, chloroform, and combinations thereof.

19. A method of reducing the concentration of volatile organic compounds in air comprising contacting the transgenic houseplant of any one of claims 1-17 with air comprising volatile organic compound vapors.

20. The method of claim 19, wherein the volatile organic compound is selected from the group consisting of: 1, 4-dichlorobenzene, benzene, 1, 3-butadiene, formaldehyde, acetaldehyde, naphthalene, acrylonitrile, carbon tetrachloride, dichlorobromomethane, dibromochloromethane, bromoform, trichloroethylene, vinyl chloride, methylchloroform, cis-1, 2-dichloroethylene, chloroform, and combinations thereof.

21. The method of claim 19 wherein the protein is cytochrome P4502E 1 or a variant or homolog thereof having at least about 90% amino acid sequence homology thereto, and the volatile organic compound is chloroform, carbon tetrachloride, benzene, or a combination thereof.

22. An air purifying biofilter, comprising: (a) a planting structure comprising one or more transgenic houseplants according to any one of claims 1-18, and (b) an air flow device coupled to the planting structure and adapted to maintain air flow around the plant.

23. The air purifying biofilter of claim 22, wherein the air flow device increases mass air flow over the foliage of the one or more transgenic house plants.

24. An air purifying biofilter according to claim 22 or claim 23, further comprising means for self-watering.

25. The air purifying biological filter of any one of claims 22-24, further comprising a light source.

26. The air purifying biofilter of any one of claims 22-25, wherein in a room with air exchange per hour with an exterior space equal to one room volume air change, the biofilter is capable of reducing particulates in the room by about 80% or more.

27. The air purifying biofilter according to any one of claims 22-26, wherein substantially all particles having an average size of 2 μm or more are removed from the air during passage through the air purifying biofilter.

28. The air purifying biological filter of any one of claims 22-27, wherein the air purifying biological filter comprises an enclosure surrounding the one or more transgenic houseplants.

29. A green wall comprising one or more transgenic houseplants of any one of claims 1-18.

30. The green wall of claim 29, further comprising an air flow device coupled to the green wall and adapted to maintain an air flow around the one or more transgenic houseplants.

Technical Field

The present invention relates to transgenic houseplants capable of removing Volatile Organic Carcinogens (VOCs) from the indoor air and biofilters comprising such transgenic plants.

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional patent application No. 62/792,726 filed on 2019, month 1 and 15, which is incorporated herein by reference in its entirety.

Statement regarding sequence listing

The sequence listing associated with this application is provided in textual format in place of the paper copy and is incorporated by reference into the specification herein. The name of the text file containing the sequence list is 70586_ Seq _ Final _2019-12-16. txt. The text file is 6.92 KB; created in 2019, 12, 16; and is being submitted with the specification through the EFS-Web.

Statement of government permission

The invention was made with government support under grant CBET-1438266 awarded by the national science foundation of the United states and grant number 2P42-ES004696-19 of the national institute of environmental health sciences. The government has certain rights in this invention.

Background

Home air is more polluted than office and school air, and those who spend most of their time at home, such as children and those who work at home, receive proportionally higher doses of home carcinogens than the general population. Infants are particularly susceptible to indoor air contamination because of their light weight and continued exposure to indoor air. The most highly-risky Volatile Organic Carcinogens (VOCs) are benzene, formaldehyde, 1, 3-butadiene, carbon tetrachloride, acetaldehyde, 1, 4-dichlorobenzene (PDCB), naphthalene, perchloroethylene, chloroform, and ethylene dichloride. VOCs that exceed acute exposure standards are acrolein and formaldehyde (during cooking) and chloroform (during showering).

Some sources of these chemicals may be eliminated or reduced. PDCB can be greatly reduced, for example, by eliminating the product containing it from home. Formaldehyde in home air can be reduced by changing the composition of building and upholstery materials, but formaldehyde is also emitted from other sources (including cooking) that are not easily eliminated. Other carcinogens from a variety of sources, such as benzene, are more difficult to eliminate and originate from fuel, outdoor air and ambient tobacco smoke stored in an attached garage.

Physicochemical methods of VOC removal include adsorption onto activated carbon, activated alumina, zeolites or other surfaces and photocatalytic oxidation. Adsorption methods are less suitable for formaldehyde and other polar compounds. Low molecular weight compounds may desorb in competition with higher molecular weight contaminants. The adsorption process is not destructive and the adsorbent must be regenerated periodically, usually remotely using energy intensive methods. The low temperature in situ process achieves energy efficient regeneration but requires external piping. Oxidation processes use photocatalytic redox to destroy catalytic materials (such as TiO)₂) The above VOC. Photocatalytic oxidation can completely mineralize most contaminants, but is not effective for chlorinated VOCs (such as chloroform). In addition, photocatalytic oxidation methods can introduce ozone into the home air, and they are energy intensive.

Indoor plants are widely touted to be able to remove air pollutants from indoor air. The method is called 'green liver' concept and is a central idea in the field of plant repair, and the plant repair is to remove foreign biological pollutants in the environment by using plants. Early studies on air detoxification of houseplants found that formaldehyde was removed from the air in a room containing chlorophytum comosum. Other researchers reported that soil or water alone could explain this removal. Subsequently, experiments with control pure cultured plants showed that plants can absorb and metabolize formaldehyde from the air. However, the formaldehyde uptake rate through foliage of typical house plants does not appear to be sufficient to remove formaldehyde from a typical room without excess plants. Several studies found that common plants can remove VOCs (such as formaldehyde and benzene) from air, but these studies yielded highly variable estimates of the rate at which specific plant species remove a given pollutant from air. The concentrations used in these tests are orders of magnitude higher than typical home air concentrations (e.g., 1-7 μ g m)^-3)。

Despite these conflicting data, plants do have many attractive features as a platform for the metabolism of organic pollutants. Unlike most bacteria, cultivated plants have excess energy to support co-metabolic catalysis. Plants have a high surface area and promote mass transfer of trace amounts of gas from the air. Plants are self-sustaining and do not require the high maintenance typical of bacterial systems. The genetic and enzymatic composition of the cultivated plants is determined in comparison to the soil bacterial community.

Plants were genetically engineered to overexpress native plant formaldehyde dehydrogenase activity, but formaldehyde removal was only 25% higher than that of non-engineered plants. Expression of the formaldehyde dehydrogenase faldh transgene from Brevibacillus brevis (Brevibacillus brevis) in transformed tobacco plants increased formaldehyde removal by a factor of three. However, to date, detoxification genes have not been expressed in house plants.

Therefore, there is a need for plants that can be grown indoors as house plants and that perform effective phytoremediation of the indoor air by metabolizing VOCs.

Disclosure of Invention

This summary is provided to present an option to the concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In some embodiments, the expression cassette further comprises a second polynucleotide sequence encoding a visual selection marker, a third polynucleotide sequence encoding a positive selection marker, a fourth polynucleotide encoding an aldehyde dehydrogenase, or a combination thereof. In some embodiments, each of the first, second, third and fourth polynucleotide sequences is operably linked to a suitable promoter functional in a transgenic indoor plant.

In some embodiments, the transgenic house plant is a transgenic scindapsus aureus (Epipremnum aureum).

In some embodiments, the mammalian cytochrome protein is a cytochrome P450 or a variant thereof or a homolog thereof having at least about 90% amino acid sequence homology thereto, such as rabbit cytochrome P4502E 1 pigment P4502E 1 or a variant thereof or a homolog thereof having at least about 90% amino acid sequence homology thereto.

In some embodiments, each of the first, second, third, and fourth polynucleotide sequences is codon optimized for a plant (e.g., a monocot).

In some embodiments, the visual selectable marker is a fluorescent protein, such as a gfp-ER, eGFP, or a variant or homolog thereof having at least about 90% amino acid sequence homology thereto. In some embodiments, the positive selection marker is hygromycin B phosphotransferase or a variant thereof or a homolog thereof having at least about 90% amino acid sequence homology thereto. In some embodiments, the fourth polynucleotide sequence encodes an aldehyde dehydrogenase such as formaldehyde dehydrogenase or a variant thereof or a homolog thereof having at least about 90% amino acid sequence homology thereto.

In some embodiments, the transgenic houseplant is capable of removing a volatile organic compound selected from the group consisting of: 1, 4-dichlorobenzene, benzene, 1, 3-butadiene, formaldehyde, acetaldehyde, naphthalene, acrylonitrile, carbon tetrachloride, dichlorobromomethane, dibromochloromethane, bromoform, trichloroethylene, vinyl chloride, methylchloroform, cis-1, 2-dichloroethylene, chloroform, and combinations thereof.

In a second aspect, provided herein is a method of phytoremediation comprising contacting a transgenic houseplant disclosed herein with air comprising volatile organic compound vapors, thereby removing the volatile organic compounds from the air. In some embodiments, contacting air comprising Volatile Organic Compound (VOC) vapors with the transgenic houseplants disclosed herein results in a reduction in the concentration of VOC in the air.

In a third aspect, provided herein is an air purifying biofilter comprising one or more of the transgenic houseplants disclosed herein. In some embodiments, an air purifying biofilter comprises: (a) a growing structure comprising one or more of the transgenic houseplants disclosed herein, and (b) an air flow device coupled to the growing structure and adapted to maintain air flow around the plant. In some embodiments, the planting structure is a green wall. In some embodiments, the air purifying biofilter system further comprises an enclosure surrounding the one or more transgenic houseplants. In some embodiments, the air purifying biofilter further comprises a device for self-irrigation, a light source, or a combination thereof.

Drawings

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the structure of binary vector pRCS2-2E1-EGFP used for transformation of scindapsus aureus (pothos ivy). In this figure, T35s is the terminator of the CaMV 35s gene; hpt is the hygromycin phosphotransferase gene, providing hygromycin resistance; OsActin is the promoter of rice (Oryza sativa) actin gene; tms is the terminator of the manopine synthase gene; 2E1 is the rabbit cytochrome P4502E 1 gene; ZmUbi is the promoter of maize (Zea mays) ubiquitin; PvUbi is the promoter of the ubiquitin gene of switchgrass (Panicum virgatum); egfp is enhanced green fluorescent protein; trbc is the terminator of the rubisco small subunit gene; LB is the left border of the T-DNA region; RB is the right border of the T-DNA region.

FIGS. 2A-2D show the transformation of scindapsus aureus by Agrobacterium (Agrobacterium) infection with the 2e1 gene. The leaf disc and petiole fragments of scindapsus aureus were infected with EHA105 harboring pRCS2-2E 1-EGFP. The explant is 20mg L^-1Hygromycin was cultured on somatic embryo induction medium for selection. (2A) Callus developed from explants after 3-4 months of selection. (2B) Hygromycin-resistant calli were transferred to a 20mg L donor^-1The hygromycin is used for inducing new seedling development in a regeneration culture medium for 2-4 months. (2C) Transferring the regenerated plant to a medium containing 20mg of L^-1Hygromycin for rooting and growth in MS medium. (2D) The PCR and RT-PCR positive transformed plants were cultured on a hygromycin-containing callus induction medium for callus induction and propagation.

FIG. 3 is a graph of transcript abundance on a scindapsus aureus strain transformed with 2e1 and egfp genes measured using quantitative RT PCR. The y-axis shows values normalized to the scindapsus aureus 5.8s rRNA gene and relative to VD1 level (n ═ 3 ± SE). The letters indicate a meaning without significant difference (p ═ 0.05, ANOVA).

Fig. 4A and 4B show EGFP signal in epidermal cells of scindapsus aureus clone VD3 observed using a fluorescence microscope. An EGFP green fluorescent signal was observed in the cytoplasm of leaf epidermal cells of scindapsus aureus clone VD3 (4A). The emission of wild type scindapsus aureus (4B) was due to autofluorescence.

FIG. 5 is a graph of the absorption of benzene by 2e 1-egfp-converted scindapsus aureus grown in liquid culture. Headspace benzene concentrations during 8 days of culture for VD3(2e1), wild type plants (WT) and No Plant Control (NPC) are shown. N is 4. Mean. + -. SE.

FIG. 6 is a graph of the uptake of chloroform by 2e1-egfp transformed scindapsus aureus grown in liquid culture. Headspace chloroform concentrations during 11 days of culture for VD3, wild type plants (WT) and No Plant Control (NPC) are shown. N is 4 ± SE.

FIG. 7 is a semilogarithmic graph of the time course of benzene concentration in the scindapsus aureus transgenic clone VD3 batch culture. R²0.781. Slope-0.249 d^-1T statistic-7.81, p-5 × 10^-7。

FIG. 8 is a semilogarithmic graph of the time course of benzene concentration in batch culture of wild type scindapsus aureus. R²0.299. Slope-0.044 d^-1T statistic-2.69 and p-0.015.

FIG. 9 is a semilogarithmic graph of the time course of chloroform concentration in batch culture of the transgenic clone scindapsus aureus VD 3. R²0.77. Slope-0.578 d^-1T statistic-5.49 and p-0.0004.

FIG. 10 is a semilogarithmic graph of the time course of chloroform concentration in batch culture of wild type scindapsus aureus. R²0.23. Slope-0.0.026 d^-1T statistic-1.32 and p-0.22.

FIG. 11 shows the sequence of an exemplary mammalian cytochrome used to make an exemplary transgenic houseplant (SEQ ID NO: 15).

Fig. 12 is a photograph of an exemplary air purifying biofilter.

FIG. 13 shows the sequence encoding an exemplary FALDH that can be used to make exemplary transgenic house plants (SEQ ID NO: 16).

FIG. 14 shows the sequence of an exemplary FALDH protein (SEQ ID NO: 18).

FIG. 15 shows the sequence (SEQ ID NO:16) encoding an exemplary ALDH1a1 useful for making exemplary transgenic house plants.

FIG. 16 shows the sequence of an exemplary ALDH1a1 protein (SEQ ID NO: 18).

Detailed Description

The present disclosure relates to genetically modified houseplants, houseplant parts, and houseplant cells and their use in the bioremediation of Volatile Organic Carcinogens (VOCs). In one aspect, provided herein are genetically modified houseplants, i.e., transgenic houseplants, capable of reducing the levels of VOCs (such as formaldehyde, benzene, and chloroform) in the surrounding air. The plants express a detoxifying transgene, e.g., mammalian cytochrome P4502 e, and have sufficient detoxifying activity against volatile organic carcinogens (such as benzene and chloroform).

In some embodiments, a transgenic houseplant disclosed herein is stably transformed with an expression cassette comprising a first polynucleotide sequence encoding a mammalian cytochrome protein, wherein the houseplant is capable of removing volatile organic compounds, such as carcinogens, from the air. As used herein, the terms "genetically engineered" and "transgenic" are used interchangeably. In some embodiments, the mammalian cytochrome protein is a cytochrome P450, a variant thereof, or a homolog thereof having at least about 80%, at least about 85%, or at least about 90% amino acid sequence homology thereto. In certain embodiments, the mammalian cytochrome protein is cytochrome P4502E 1.

Mammalian cytochrome P4502E 1 (also referred to herein as 2E1) can oxidize a wide range of important VOCs such as benzene, chloroform, trichloroethylene, and carbon tetrachloride that can be found in typical household air. CYP2e1(2e1) genes have previously been successfully introduced into trees, but such modifications to house plants have not been proposed. Any mammalian cytochrome P4502E 1 can be used to produce a houseplant disclosed herein, e.g., rabbit cytochrome P4502E 1. In some embodiments, the mammalian cytochrome has a sequence as shown in fig. 11, a variant thereof, or a homolog thereof having at least about 80%, at least about 85%, or at least about 90% amino acid sequence homology thereto. In some embodiments, the mammalian cytochrome comprises SEQ ID NO:15, a variant thereof, or a homolog thereof having at least about 80%, at least about 85%, or at least about 90% amino acid sequence homology thereto.

In some embodiments, the polynucleotide sequence encoding the mammalian cytochrome protein (e.g., rabbit cytochrome P4502E 1) is codon optimized for a plant. In certain embodiments, the polynucleotide sequence encoding the mammalian cytochrome protein is codon optimized for monocots. In some embodiments, the first polynucleotide sequence encoding a mammalian cytochrome is operably linked to a first promoter that is functional in a houseplant. Any suitable promoter that is functional in a houseplant disclosed herein can be used in a transgenic houseplant disclosed herein. In some embodiments, the first promoter is a promoter functional in scindapsus aureus (Epipremnum aureum). In certain embodiments, the first promoter is a promoter disclosed by Zhao et al (Zhao, j.t.; Li, z.j.t.; Cui, j.; Henny, r.j.; Gray, d.j.; Xie, j.h.; Chen, j.j., Efficient genetic engineering and Agrobacterium-mediated transformation of pothos (Epipremnum aurum) 'Jade' Plant tissue Org 2013,114, (2), 237-. Exemplary promoters suitable for inclusion in the expression cassettes of the transgenic housekeeping plants disclosed herein are described in the examples below.

In some embodiments, the houseplant is scindapsus aureus (Epipremnum aureum), commonly known as scindapsus aureus (pothos ivy). Other common names include bergamot (pothos), ivy (devil's ivy), and variegated vine (variegated philiodendron). Scindapsus aureus offers several advantages over other houseplants or other plants used to produce the transgenic houseplants disclosed herein: it is vigorous, viable, grows well in low light, and does not flower in indoor or outdoor cultures in the united states or canada, which is an advantage with respect to biosafety concerns regarding the release of transgenic plants into the environment.

In some embodiments of the transgenic houseplants, houseplant parts, and houseplant cells disclosed herein, the expression cassette further comprises a second polynucleotide sequence encoding a visual selection marker. Any suitable visual selection marker may be used. In certain embodiments, the visual selectable marker is a fluorescent protein. Exemplary fluorescent proteins suitable for inclusion as visual markers include green fluorescent proteins, such as mGFP-ER and eGFP, variants thereof, and homologs thereof having at least about 80%, at least about 85%, or at least about 90% amino acid sequence homology thereto.

The inclusion of a visual selection marker enables the differentiation of plants from wild type, thereby providing additional biosafety assurance by observing the unique fluorescent characteristics of the marker. Any suitable visualization method may be used. For example, the transgenic plants disclosed herein can be monitored by simple visual observation or by using a detection device. Instruments and methods for detecting and quantifying visual markers, such as fluorescent Proteins, in Plants are known in the art, for example, the instruments and methods disclosed in Reginal J.Millwood, Hong S.Moon, and C.New Stewart Jr.fluoro Proteins in Transgenic Plants, Reviews in Fluorescence, 387-.

In some embodiments, the polynucleotide sequence encoding the visual marker (e.g., green fluorescent protein) is codon optimized for the plant. In certain embodiments, the second polynucleotide sequence encoding the visual marker is codon optimized for monocots. In some embodiments, the second polynucleotide sequence is operably linked to a second promoter functional in a houseplant. Any suitable promoter functional in the transgenic houseplants disclosed herein (such as those described above) may be used in the transgenic houseplants disclosed herein.

In certain embodiments, the expression cassette further comprises a third polynucleotide sequence encoding a positive selection marker. A positive selection marker is a gene that confers resistance to an agent that kills wild-type houseplants. Any suitable positive selection marker may be used. For example, in certain embodiments, the positive selection marker is a protein that confers antibiotic resistance to an indoor plant. In certain embodiments, the positive selection marker is hygromycin B phosphotransferase, which confers resistance to hygromycin to houseplants (e.g., scindapsus aureus). In other embodiments, the positive selection marker is a gene that confers resistance to herbicides, for example, hompson CJ, Movva NR, Tizard R, et al, characterisation of the herbicide-resistance gene bar from microorganisms. 6(9) 2519-23, the disclosure of which is incorporated herein by reference.

In some embodiments, the polynucleotide sequence encoding the positive selection marker (e.g., hygromycin B phosphotransferase) is codon optimized for a plant (e.g., a monocot). In some embodiments, the third polynucleotide sequence is operably linked to a third promoter functional in a houseplant (such as the promoters described above).

In some embodiments, it is advantageous to have transgenic houseplants that express more than one detoxification enzyme capable of removing VOCs from the home air, e.g., for removing more than one chemical class of VOCs. Thus, in some embodiments, the transgenic houseplant described herein expresses a mammalian cytochrome (such as 2e1) and one or more other detoxification genes. For example, as described in the examples below, the faldh gene may be overlaid with 2e1 and/or other detoxification genes in a vector used to genetically modify house plants (such as scindapsus aureus) to produce house plants that can degrade formaldehyde, which is one of the most important indoor air VOCs. Thus, in certain embodiments of the transgenic houseplant, houseplant part or houseplant cell disclosed herein, the expression cassette further comprises a fourth polynucleotide sequence encoding formaldehyde dehydrogenase, a variant thereof, or a homologue thereof having at least about 80%, at least about 85%, or at least about 90% amino acid sequence homology thereto. In some embodiments, the fourth polynucleotide sequence is operably linked to a fourth promoter functional in a houseplant. Any suitable promoter functional in the transgenic houseplants disclosed herein (such as those described above) may be used in the transgenic houseplants disclosed herein.

In some embodiments, provided herein is a houseplant cell or a houseplant part stably transformed with an expression cassette comprising a first polynucleotide sequence encoding a mammalian cytochrome protein operably linked to a first promoter functional in a houseplant and a second polynucleotide sequence encoding a visual selectable marker operably linked to a second promoter functional in a houseplant.

In yet another aspect, disclosed herein is a method of phytoremediation comprising contacting a transgenic houseplant of the present disclosure with air comprising volatile organic compound vapors or volatile organic carcinogen vapors, thereby causing volatile organic compounds to be removed from the air. In certain embodiments, the air is room air, such as household air.

In some embodiments, the transgenic houseplants disclosed herein can remove or reduce the concentration of any volatile organic carcinogens or Volatile Organic Compounds (VOCs) in the air that can be oxidized by mammalian cytochromes. As used herein, the term "removing" a contaminant (such as a VOC) includes partial removal, e.g., a reduction in concentration, and complete removal, e.g., below levels detectable by methods commonly used to analyze such VOCs. In some embodiments, VOCs suitable for removal using the methods disclosed herein include aromatics, unsaturated hydrocarbons, ketones, aldehydes, and halogenated hydrocarbons. Non-limiting examples of such VOCs include 1, 4-dichlorobenzene, benzene, 1, 3-butadiene, acetaldehyde, naphthalene, acrylonitrile, carbon tetrachloride, dichlorobromomethane, dibromochloromethane, bromoform, trichloroethylene, vinyl chloride, methylchloroform, cis-1, 2-dichloroethylene, chloroform, and combinations thereof.

In yet another embodiment, provided herein is an air purifying biofilter comprising one or more transgenic houseplants of the present disclosure, e.g., stably transformed with an expression cassette comprising a polynucleotide sequence encoding a mammalian cytochrome described above. In some embodiments, the transgenic plant is scindapsus aureus stably transformed with an expression cassette comprising a polynucleotide sequence encoding a mammalian cytochrome described above.

To maximize the phytoremediation potential of the transgenic houseplants disclosed herein, in some embodiments, it is advantageous to construct or increase air mass transfer around the transgenic houseplants, e.g., moving air over the foliage of the houseplants disclosed herein. In certain embodiments, the air purifying biofilter further comprises means for increasing the mass air flow above the foliage of the transgenic houseplant, i.e., an air flow device. In some embodiments, an air purifying biofilter comprises: (a) a plant container comprising one or more of the transgenic houseplants disclosed herein, and (2) an air flow device coupled to the plant container and adapted to maintain an air flow around the plant.

Any suitable means for increasing the mass air flow may be included in the air purification units or biofilters disclosed herein, including mechanical means (such as by operating an electric or manual fan). In some embodiments, the device may generate a pressure or temperature differential, and the increase in air flow may occur passively, for example, as a function of a pressure differential or temperature gradient present in the environment surrounding the transgenic houseplant.

In some embodiments, the air purification unit comprises an enclosure housing one or more transgenic houseplants. In some embodiments, the air purification unit is a green wall comprising one or more transgenic houseplants of the present disclosure and a means for increasing the mass air flow around the one or more plants. In some embodiments, a green wall is a vertical building structure that is purposefully planted covered, and may also be referred to as a living wall or vertical garden.

In some embodiments, the biofilter complies with standards used by the home appliance manufacturers association (AHAM), for example, the rating standards for particulate biofilters or "2/3 rules": i.e. assuming a ceiling of 8ft, clean air delivery rate (CADR, ft)³Min or m³H) greater than or equal to the room area ft²2/3 times higher. Particulate filters were tested by AHAM in a standard room containing 1008ft³(28.5m³) 8ft ceiling and 126ft area²(11.7m²). CA for filters meeting the 2/3 rule for this roomDR will equal 2/3 times 126 or 84cfm (2.4 m)³Min) or 0.67cfm CADR/ft²。

Thus, in some embodiments, the biofilters disclosed herein are capable of reducing particulates in a room by about 75% or more, about 80% or more, about 85% or more, or about 90% or more, the room exchanging air with the exterior space per hour equaling the air change of one room volume. In some embodiments, substantially all particles having an average size of 2 μm or greater are removed from the air during passage through the air purifying biofilter.

The following examples are provided to illustrate, but not to limit, the present invention.

Examples

Materials and methods

Preparation of scindapsus aureus

Golden kudzuvine (Golden pothos ivy) plant obtained from retail gardening store at 50 mu E m^-2sec^-1The plants were grown under light in a plant room at 25 ℃ with a 16 hour day/8 hour night cycle. The stem sections were cut, surface sterilized with 15% sodium hypochlorite, and then washed 3 times with sterile deionized water. The sterilized stem segments are cultured on solid Murashige and Skoog's (MS) basal medium 25 in a culture vessel. After 1-2 months of culture in the light, new leaves and roots grow out of the stem and these sterile plants are used for genetic engineering by infection with engineered agrobacteria.

Vector construction and Gene transformation

For genetic engineering of scindapsus aureus, gene vectors were constructed containing transgenes 2e1, egfp and hpt, each flanked by promoter and terminator sequences appropriate for scindapsus aureus. The hpt gene encodes hygromycin B phosphotransferase, which confers hygromycin resistance. Hygromycin is used to select for transformed cells because it kills wild-type scindapsus aureus. These three genes were integrated into a transformation vector ("binary vector") based on a cloning vector system called pSAT containing an insertion site for use with specific restriction enzymes (Chung, S.M.; Frankman, E.L.; Tzfira, T., A versatile vector system for multiple gene expression in plants. trends in Plant Science 2005,10, (8), 357-361). The binary vector was then introduced into the engineered agrobacterium strain EHA105 for infection of scindapsus aureus callus cultures.

The rabbit cytochrome P4502 e1 gene was amplified by Polymerase Chain Reaction (PCR) from plasmid pSLD50-6 (primer sequences, SEQ ID NOs 1-14 listed in table 1) that was gifted by s.l. doty (university of washington) and double digested with restriction enzymes HindIII and KpnI.

TABLE S1 DNA sequences of primers used to prepare exemplary vectors

The 2E1 DNA was then inserted into the cloning vector pNSAT3a to produce pNSAT3a-2E 1. Following insertion of pNSAT3a, the 2e1 gene was integrated between the promoter and terminator sequences to produce an expression cassette that drives expression of 2e1 in plant cells. The EGFP gene was cloned by PCR from vector pGH00.0126 (as described in Maximova, S.et al, Stable transformation of the same organism cacao L.and flexibility of the matrix attachment regions on GFP expression. plant Cell Rep 2003,21, (9), 872-83) and inserted into pNSAT6a as a HindIII-PstI fragment to yield pNSAT6 a-EGFP. The Expression cassettes for the HPT, 2E1 and EGFP genes were excised from pNSAT1a-HPT using the restriction enzymes AscI, I-PpoI and PI-PspI, respectively (as described for Zhang, L.; et al, Expression in grams of multiple variants for the deletion of variants on live-fire variants. plant Biotechnol J2017, 15, (5), 624) and pNSAT3a-2E1 and pNSAT6a-EGFP vector were excised and inserted into pRCS2 binary vector to produce pRCS2-2E 1-EGFP.

Binary vector pRCS2-2E1-EGFP was transferred into Agrobacterium strain EHA105 by freeze-thaw methods (Chen, H.; Nelson, R.S.; Sherwood, J.L., Enhanced recovery of transformations of Agrobacterium tumefaciens after-freeze-transformation and drug selection Biotechniques 1994,16, (4),664-8,670) and the resulting strain EHA105(pRCS2-2E1-EGFP) contained 50mg L^-1Rifampicin, 100mg L^-1Spectinomycin and300mg L^-1streptomycin was grown in LB medium (lysogen broth) for infection with scindapsus aureus. EHA105(pRCS2-2E1-EGFP) started at 50mg L^-1Rifampicin and 100mg L^-1Spectinomycin in 100mL LB medium and cultured overnight at 28 ℃ on a rotary shaker at 200 rpm. The bacteria were centrifuged at 4,000rpm for 10min and resuspended in liquid E medium (containing 2mg L) containing 100. mu.M Acetosyringone (AS)^-1Thiadiazoluron (TDZ) and 0.2mg L^-1MS medium of 1-naphthylacetic acid (NAA) and cultured under the same conditions until the OD600 (absorbance of bacterial suspension at 600 nm) reaches 0.8-1.0.

The following methods for transforming scindapsus aureus are adapted from ZHao et al (ZHao, J.T., et al, effective genetic organization and Agrobacterium-mediated transformation of pots (Epipremnum aureum) 'Jade'. Leaf discs and petiole segments from sterile scindapsus aureus plants were immersed in agrobacterium culture at 25 ℃ for 20 minutes, then transferred to double-layered filter paper wetted in petri dishes with liquid E medium containing 100 μ M AS, and co-cultured at 25 ℃ for 5 days. Leaf discs and petiole fragments were washed with sterile water and transferred to a medium containing 100mg L^-1Cefotaxime, 100mg L^-1Carbenicillin (Phytotechnology Laboratories) and 20mg L^-1Hygromycin was screened in E medium. Explants were subcultured to fresh selection medium every three weeks.

After 2-3 months of selection, somatic embryos developed from explants on selection medium were transferred to fresh medium for another one month and then transferred to medium containing 100mg L^-1Cefotaxime, 100mg L^-1Carbenicillin and 20mg L^-1Hygromycin G Medium (2 mg L)^-16-benzylaminopurine (6-BA) and 0.2mg L^-1MS medium of NAA) and cultured under light for regeneration. After two months of culture, the regenerated seedlings were transferred to a medium containing 100mg L^-1Cefotaxime, 100mg L^-1Carbenicillin and 20mg L^-1Hygromycin in MS medium for rooting and growth, which requires two more months of culture.

Molecular analysis of transformed plants

For Polymerase Chain Reaction (PCR) analysis, DNA was purified from hygromycin resistant plants using DNeasy plant mini kit (Qiagen, Valencia, CA, USA). The PCR reaction was performed using 2e1 and egfp cassette specific primers (as shown in table 1).

Total RNA was extracted from leaves of plants using RNeasy plant mini kit (Qiagen, Valencia, CA, USA). For real-time quantitative RT-PCR analysis, 1. mu.g of total RNA was transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madison, Wis., USA). Real-time quantitative PCR was performed on a fluorescent thermocycler Light Cycler (Roche) using the SensiFAST SYBR No-ROX kit (Bioline, Memphis, TN, USA), and data were analyzed using Light Cycler 3 software (Roche). A standard curve was constructed from plasmid DNA of prcs2-2e 1-egfp. Transcript values measured using RT-qPCR were normalized to the scindapsus aureus 5.8S gene and presented relative to the transcript levels in clone VD 1.

Absorption of benzene and chloroform by the transformed scindapsus aureus

Sterile seedlings of scindapsus aureus (1g) were subjected to septum valveValco Instruments Co.Inc., 614163) blocked and incubated in 40mL Volatile Organic Assay (VOA) vials (Fisher Scientific, 14-823-213) containing 5mL of half-strength Hoagland solution (Caisson Labs, HOP01-10 LT.1). Wild type untransformed seedlings and no plant controls were grown in parallel with clone VD3, in four replicates per treatment.

Benzene gas was injected into the vial using a gas-tight glass syringe to achieve 1850. + -. 160mg m^-3According to henry's law, gas-liquid distribution is considered. The vials were incubated for 9 days with rotary shaking at 80 rpm. Benzene concentration was determined by manually injecting 100 μ L headspace into GC-FID (flame ionization detector) (Perkin Elmer automation system XL). The chromatographic parameters are as follows: column box temperature 60 deg.C, sample injector temperature 250 deg.C, detector temperature 250 deg.C, 1.33ml min^-1Nitrogen carrier gas, using ResTek RTX-1 microcapillary column (ResTek, 10121).

Similarly, chloroform was introduced into VOA vials from a sealed aqueous dilution of chloroform (Acros Organics, 423550010) using a gas-tight glass syringe. Transformed seedlings, Wild Type (WT) and no plant controls were replicated in quadruplicate and headspace samples were taken for analysis of chloroform levels by gas chromatography with electron capture detector (GC-ECD) (Perkin Elmer AutoSystems XL) and VOCOL capillary column 60m × 0.53mm (Sigma). The chromatographic parameters were: the detector temperature was 325 ℃ and the nitrogen carrier gas was 1.76ml min^-1100ml min of split flow^-1The temperature of the column box is 100 ℃, and the temperature of the sample inlet is 300 ℃.

EGFP fluorescence

EGFP signals of Epipropa scintillans were observed by fluorescence microscopy using the LSM 5PASCAL system (ZEISS). EGFP signals were excited by blue light and fluorescence was collected using a FITI filter. Pictures were taken using an Axiocam 503 monocular camera and the software ZEN 2.3 lite.

Data analysis

Data were analyzed for statistical significance using ANOVA in Microsoft Excel software (Microsoft Excel 2016 MSO). When significant differences were obtained by ANOVA analysis, Fisher Least Significant Difference (LSD) method was performed to compare the mean values. The groups with statistically significant differences (p < 0.05) are marked with letters in the figure.

Calculation of biofilter size

The biofilters of the present disclosure are designed according to the criteria used by AHAM for rating particulate biofilters (2/3 rules): that is, assuming a ceiling of 8ft, the clean air delivery rate (CADR, ft)³Min or m³H) should be greater than or equal to the room area ft²2/3 times higher. This value is based on a model that assumes a 80% reduction in particulates in a room that exchanges hourly with the exterior space equal to the air change of one room volume. Particulate filters were tested by AHAM in a standard room containing 1008ft³(28.5m³) 8ft ceiling and 126ft area²(11.7m²). For that room, the CADR of the filter that satisfies the 2/3 rule will be equalAt 2/3 times 126 or 84cfm (2.4 m)³In/min). This gives a 0.67cfm CADR/ft²。

To calculate the size and operating parameters of the GM plant biofilter, a primary model was used, which assumed that the contaminant removal rate was a function of contaminant concentration. The derivation of this model is as follows. The mass balance of the entire biofilter can be expressed as

QC_in-QC_out＝Sink，

Wherein Sink represents the removal rate of pollutants in the biofilter, mu g h^-1And are and

q-air flow through the biofilter, m³h^-1

C_inAir contaminant concentration into the biofilter, μ g m^-3

C_outConcentration of air contaminants leaving the biofilter, μ g m^-3。

Assuming that the removal of contaminants is related to contaminant concentration, proportional to plant biomass, and the biofilter is fully mixed, the concentration in the biofilter is equal to the effluent concentration.

Sink＝V_fKM_pC_outWherein

V_fVolume of biofilter, m³

K is the first order kinetic rate constant, h^-1(g plant Biomass)^-1

M_pPlant biomass, g

Therefore, the temperature of the molten metal is controlled,

Q(C_in-C_out)＝V_fKM_pC_out

definition of

E-the removal efficiency in one pass through the biofilter,

thus, the concentration time course in batch experiments of uptake of benzene and chloroform by transgenic plants of the disclosure, such as the exemplary scindapsus aureus clone vD3, is exponentially distributed.

Calculation of first order rate constants

In the biofilter size calculations, chloroform was chosen instead of benzene absorption because benzene was oxidized by 2E1 to phenol, which may be toxic to plants at the concentrations used in these experiments. Almost immediate oxidation of chloroform to the non-toxic product CO₂And chloride ions; thus, the rate of chloroform is believed to be more representative of the rate at which both contaminants are visible at the home level.

The level of 2e1 gene expression in transformed scindapsus aureus was independent of benzene or chloroform concentration. Thus, the kinetic parameters of contaminant degradation are expected to remain constant with contaminant concentration.

Biofilter design using exemplary scindapsus aureus VD3

Contaminant uptake rate was determined in 40mL vials containing plants weighing about 1 g. The first order rate of chloroform absorption by VD3 scindapsus aureus is shown in table 2. These rates were used for the exemplary biofilter design. This is a conservative assumption, since the absorption rate of forced air biofilters may be greater than in 40mL vials due to convection and turbulence in the biofilter. An exemplary scindapsus aureus VD3 has a first order rate constant of 0.522d for chloroform removal^-1(g Biomass)^-1(as shown in table 2).

TABLE 2 first order kinetic constants for the gene modification and chloroform uptake by wild type scindapsus aureus plants normalized to plant biomass and leaf area

The slope of the semilogarithmic plot of the time course of wild-type chloroform uptake was not significantly different from the zero line (p 0.22).

If the air flow rate of the selected biofilter is equal to 1150cfm (333 m)³H) and a plant mass of 11kg (24lb), 1m is used³(35.3ft²) Can reach a CADR design of 84 cfm. Normalized to room area and assuming a plant density of 11kg/m in the biofilter³(0.7lb/ft³) Will adopt 0.052m³(1.8ft³) And 0.57kg (1.25lb) of plant biomass to provide 0.67cfm CADR/ft²Or 0.91kg of plant biomass/m²。

The leaf area of scindapsus aureus per g biomass is about 29cm²Thus, the leaf area of 11kg of plants was 32m². The average area of the epipremnum aureum leaves grown indoors per leaf (2 surfaces) is about 64cm². The indoor grown mature scindapsus aureus grew to about 10m with leaves about every 2cm, so there were about 500 leaves per mature plant, and the total surface area of each plant was 3.2m²The biomass of each mature plant was about 1.1 kg. Thus, 11kg of plant biomass equates to about 10 mature scindapsus aureus plants, a number that can be easily achieved in an exemplary biofilter (e.g., green wall).

Results

Vector construction and generation of transgenic scindapsus aureus

The structure of the plasmid pRCS2-2E1-EGFP for transforming scindapsus aureus is shown in figure 1. To achieve constant, high-level expression, all transgenes were driven by constitutive monocot promoters. The hygromycin resistance gene hpt is driven by the actin promoter of rice (Oryza sativa) (McElroy, D.et al, Isolation of an effective Activity in promoter for use in rice transformation. the Plant cell 1990,2, (2),163-71), the 2e1 gene is driven by the ubiquitin promoter of maize (Zea mays) (Cornejo, M.J.; et al, Activity of a main gene in transport. Plant molecular biology 1993,23, (3),567-81), and the egfp gene is driven by the ubiquitin promoter of switchgrass (Panicum virgatum) (Man, D.G.; et al, Gateway-soluble genes for high-throughput gene, Plant cell 13, 2, 36).

Explants of scindapsus aureus were infected with EHA105 containing vector pRCS2-2E1-EGFP and screened on callus induction medium with hygromycin as selection agent for 2-3 months. A trichome cell embryo that developed from the cut edges of the leaf disc and petiole segments (fig. 2A). During subsequent culturing, callus is formed at the base and more clustered somatic embryos develop from the callus. After 3-4 months of culture, hygromycin resistant calli were transferred to regeneration medium to induce seedlings. After further culturing for 2-3 months, seedlings with shoots and roots develop from the somatic embryos. Some seedlings developed shoots only (fig. 2B). These plants were transferred to MS medium containing hygromycin for further growth and rooting (FIG. 2C). Leaf discs of PCR and RT-qPCR positive lines containing 15mg L^-1Hygromycin were cultured on E medium to induce somatic embryo propagation while selection was still being performed (fig. 2D).

Molecular analysis to confirm transformation of hygromycin-resistant strains

Integration of the target gene into the genome of scindapsus aureus was confirmed by PCR triggered by primer pairs annealing to the promoter and terminator regions of the 2e1 and egfp cassettes (data not shown). To measure the transcriptional abundance of the 2e1 and egfp genes, RT-qPCR was performed on eight transgenic lines VD1-VD 8. egfp expression levels were below 2e1 and were divided into two groups with significant differences between VD3 and VD2 or VD7 (p < 0.01, fig. 3). The expression level of the 2e1 gene was significantly different between different transformation lines (p ═ 0.00001). The clonal lines VD3, VD7 and VD8 had higher expression levels of 2e1 than the other lines. The non-transformed clonal lines have observable changes in morphology or growth compared to the wild type.

EGFP Observation

Using a fluorescence microscope, EGFP fluorescence was observed near the plasma membrane and around the nucleus due to the presence of vacuoles in epidermal cells of scindapsus aureus leaves (fig. 4A). The emitted light is slightly greater than that of the wild type, but weaker. Wild-type cells are generally weakly autofluorescent, but not specifically from the cytoplasm. There was no visible green fluorescence in the transformed scindapsus aureus under hand-held UV lamp illumination.

Absorption of benzene by converted scindapsus aureus

To determine the ability of 2e 1-egfp-converted scindapsus aureus to absorb benzene, plants were incubated with VOCs in closed vials. Benzene (144 μ g) was injected into 40mL VOA vials containing transformed and wild type scindapsus aureus to reach 2500mg m^-3Final headspace concentration of benzene. After three days of culture, the benzene concentration in vials with the exemplary VD3 plant dropped dramatically (fig. 5). After eight days, the benzene concentration in the plant-free vials decreased by about 10%. After three days of culture, the benzene concentration in vials containing the exemplary VD3 plants was significantly different compared to vials containing wild type plants (p ═ 0.039), with p ═ 0.012 at day 4 and p ═ 0.0008 at day 8.

The time course of the benzene concentration in vials with the exemplary transformed scindapsus aureus VD3 was plotted on a semi-logarithmic axis and fitted by linear regression with a first order rate constant equal to (-0.249 d)^-1FIG. 7), or-0.115 d^-1(g fresh Biomass)^-1. The leaf area of the scindapsus aureus is 29cm²(g fresh Biomass)^-1Thus the kinetic constant is equivalent to-39.8 d^-1(m²Leaf area)^-1Normalized to the leaf area. Slope of best linear fit (-0.044 d) of semi-logarithmic plot of time course of benzene concentration in wild type plants^-1Fig. 8) had a significant difference from zero (p ═ 0.015) indicating that wild-type plants did indeed take up some benzene. Wild type scindapsus aureus absorbs benzene at a first rate normalized to biomass equivalent to-0.024 d^-1(g Biomass)^-1Or-8.5 d^-1(m²Leaf area)^-1Normalized to the leaf area. The normalized rate constant for benzene uptake by exemplary transformed clone VD3 was wild-type4.7 times.

Absorption of chloroform by an exemplary transformed scindapsus aureus

The chloroform concentration in the headspace of the vials cultured with the exemplary VD3 plant dropped rapidly, while the chloroform concentration did not change significantly with wild type seedlings and no plant control cultures (fig. 6). In vials containing the exemplary clone VD3 plant, the chloroform concentration decreased by 82% over the first 3 days, and no chloroform was detected after 6 days. Linear regression of the semi-logarithmic plot of chloroform data yielded a linear regression equal to-0.549 d^-1First order degradation constant (fig. 9). The slope of the best linear fit of the semilogarithmic curve of the time course of chloroform concentration in wild-type plants (fig. 10) was not significantly different from the zero line (p ═ 0.22), indicating that no chloroform was absorbed by wild-type plants. Exemplary VD3 normalized to biomass converted scindapsus aureus with a rate constant of 0.552d^-1(g fresh Biomass)^-1Equal to 180d^-1(m²Leaf area)^-1Normalized to the leaf area.

Discussion of the results

VOCs in indoor air pose a significant cancer risk to vulnerable groups, such as children, but there is currently no practical, sustainable technology available to remove them at home. Physicochemical processes based on adsorbents and oxidation processes are energy intensive and have limited use for removing formaldehyde and chloroform, respectively. Various houseplants are touted as having the ability to remove VOCs from the air, but the plant uptake rates vary exponentially from one study to another. Many studies appear to be affected by artificially enhancing soil bacterial activity at high VOC concentrations. Herein, in the case of benzene, high VOC concentrations were used to facilitate analysis by manual injection of headspace samples onto GC-FID, but the assay was performed under sterile conditions with no bacterial activity. As can be seen from fig. 5 and 6, there was little or no loss of benzene or chloroform in the vials containing the wild-type scindapsus aureus, while most of the benzene and all of the chloroform in the vials with the exemplary clone VD3 expressing 2E1 were removed within 6 days. These results indicate that genetically modified scindapsus aureus has an effectiveness for removing VOCs compared to wild-type scindapsus aureus.

The expression of green fluorescent protein was intended as a visual indication that scindapsus aureus was transformed, but the fluorescence of transformed clone VD3 was too weak to be seen without a microscope. Other variants of GFP, such as mGFP-ER and the use of stronger monocot promoters, may provide stronger fluorescence.

The transgenic houseplants described herein can be made to remove VOCs from the home air by expressing 2e1 in combination with other detoxification genes. Formaldehyde, another VOC that constitutes the greatest risk in household air, is of greatest concern. Overexpression of the faldh gene of Brevibacillus brevis in tobacco confers plants with high tolerance to HCHO and improves the ability to absorb formaldehyde 2-3 times faster than wild type plants. The faldh gene can be stacked with 2e1 and other detoxification genes in a vector for genetic engineering of the vine to produce plants that can degrade most important indoor air VOCs. Thus, in certain embodiments of the transgenic houseplant disclosed herein, the expression cassette further comprises a polynucleotide sequence encoding an aldehyde dehydrogenase (ALDH), such as FALDH or ALDH1a 1.

Thus, in some embodiments, a transgenic houseplant disclosed herein comprises polynucleotide sequence SEQ ID NO 16 or 18. In some embodiments, the aldehyde dehydrogenase includes the peptide sequence of SEQ ID NO 17 or 19.

Since the expression of the 2E1 gene in the transformed scindapsus aureus is under a constitutive promoter, the expression level of 2E1 is independent of benzene or chloroform concentration. Thus, kinetic parameters for contaminant degradation are expected to remain unchanged for the concentration of contaminant.

Performance of the closed forced air biofilter Using the same first order degradation constant of 0.52d as observed in chloroform batch experiments^-1(g Biomass)^-1Empirical calculations are performed. For a volume of 0.7m³Air flow rate of 300m³h^-1About 10kg of the exemplary scindapsus aureus VD3 removed 34% of the chloroform at a time. The biological filter has a Clean Air Delivery Rate (CADR) of 100m³h^-1Comparable to the CADR of current commercial in-home particulate filters. This calculation demonstrates genetically modified plants, such as hereinThe disclosed transgenic houseplant has practical utility for sustainable phytoremediation of domestic air.

Biofilters using transgenic plants have the advantages of low energy consumption and low maintenance requirements compared to current chemical/physical methods for removing VOCs from indoor air. All removal methods require means to pass air through the equipment, however, unlike the biological filters disclosed herein, the adsorption method requires a large energy expenditure to regenerate the media, while the photo-oxidation method requires a high energy input to oxidize the contaminants, making these methods less sustainable. In addition to the energy required for air flow, transgenic plant repair requires only little additional energy. Scindapsus aureus is well suited for medium and low light levels and therefore typically does not require artificial lighting, giving phytoremediation its inherent sustainability advantages.

For convenience, certain terms used in the specification, examples, and appended claims are provided herein. These definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims.

The term "or" as used in the claims and specification is intended to mean "and/or" unless explicitly indicated to refer to alternatives only or to alternatives being mutually exclusive, although the disclosure supports definitions referring to alternatives and "and/or" only.

The terms "a" and "an" when used in conjunction with the term "comprising" in the claims or the specification mean one or more, unless specifically stated otherwise.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an open-ended and inclusive sense, and not in an inclusive, exclusive or exhaustive sense. For example, the term "including" may be understood to mean "including, but not limited to". The term "consisting essentially of … …" or grammatical variants thereof means that the referenced subject matter can include additional elements not recited in the claims, but which do not materially affect the basic and novel characteristics of the claimed subject matter.

As used herein, the term "about" means a number within a slight variation above or below the stated reference value. For example, "about" may refer to a value within 10% of the stated reference value, above or below. As used herein, "plant part" refers to tissues, organs, seeds, and cut parts (e.g., cuttings) that retain the distinguishing characteristics of the parent plant.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Sequence listing

<110> university of Washington

L is sheet

S, E, Stellard

<120> degradation of indoor air pollutants by genetically modified plants

<130> UWOTL-1-70586

<150> US 62/792,726

<151> 2019-01-15

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Met Ala Val Leu Gly Ile Thr Val Ala Leu Leu Gly Trp Met Val Ile

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Leu Leu Phe Ile Ser Val Trp Lys Gln Ile His Ser Ser Trp Asn Leu

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His Gly Tyr Lys Ala Val Arg Glu Met Leu Leu Asn His Lys Asn Glu

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Ser Leu Thr Thr Leu Arg Asp Tyr Gly Met Gly Lys Gln Gly Asn Glu

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Asp Arg Ile Gln Lys Glu Ala His Phe Leu Leu Glu Glu Leu Arg Lys

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Thr Gln Gly Gln Pro Phe Asp Pro Thr Phe Val Ile Gly Cys Thr Pro

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Phe Asn Val Ile Ala Lys Ile Leu Phe Asn Asp Arg Phe Asp Tyr Lys

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Asp Lys Gln Ala Leu Arg Leu Met Ser Leu Phe Asn Glu Asn Phe Tyr

195 200 205

Leu Leu Ser Thr Pro Trp Leu Gln Val Tyr Asn Asn Phe Ser Asn Tyr

210 215 220

Leu Gln Tyr Met Pro Gly Ser His Arg Lys Val Ile Lys Asn Val Ser

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Glu Ile Lys Glu Tyr Thr Leu Ala Arg Val Lys Glu His His Lys Ser

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Leu Asp Pro Ser Cys Pro Arg Asp Phe Ile Asp Ser Leu Leu Ile Glu

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Met Glu Lys Asp Lys His Ser Thr Glu Pro Leu Tyr Thr Leu Glu Asn

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Arg Met Pro Ser Val Arg Asp Arg Val Gln Met Pro Tyr Met Asp Ala

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Val Val His Glu Ile Gln Arg Phe Ile Asp Leu Val Pro Ser Asn Leu

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Lys Gly Thr Val Val Ile Pro Thr Leu Asp Ser Leu Leu Tyr Asp Lys

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